Chanez Bennacef
email: chanez.bennacef@univ-lorraine.fr

Sylvie Desobry-Banon
email: sylvie.desobry-banon@univ-lorraine.fr

Laurent Probst

Stéphane Desobry

Advances on Alginate use for spherification to encapsulate biomolecules

Keywords: Alginate, Hydrocolloids, Spherification, Encapsulation, Composite, Coextrusion

Spherification methods to produce particles of millimetric size are detailed in this review, especially for encapsulation purpose and using alginate. Therefore, alginate properties and factors impacting its gelation, counting its structure and experimental conditions such as pH, temperature, crosslinker concentration, residence time and ionic strength, are largely discussed. Furthermore, the interest for using co-polymer in spherification is investigated. Thereby, alginate composite gels can enhance encapsulation efficiency, mechanical stability, barrier properties and thermal stability. The advantages and limitations of their use are discussed for each spherification process to improve the knowledge about alginate versatility. Alginate is still one of the most promising encapsulation materials.

1. Introduction

Alginate is the salt of alginic acid, a natural polymer of uronic acids (mannuronic and guluronic acids) extracted especially from brown marine algae (Blanco-Cabra et al., 2020; [START_REF] Boyd | Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide[END_REF][START_REF] Günter | Physicochemical and swelling properties of composite gel microparticles based on alginate and callus cultures pectins with low and high degrees of methylesterification[END_REF] and also from some bacteria, such as

Pseudomonas aeruginosa.

The alginate gelation is one common way to produce beads or capsules. The term spherification used in this review covers all the methods employed to design such particles of millimetric size, ideally spherical. In this review, we consider spherification with the aim of encapsulating different ingredients and active principles.

Alginate is Generally Recognized as Safe (GRAS), being nontoxic, nonantigenic, satisfactorily biocompatible, and sufficiently biodegradable [START_REF] Lee | Alginate: Properties and biomedical applications[END_REF]. As a result of its physicochemical properties, alginate is used for a large variety of technical applications, as a coating material in textile industry [START_REF] Misbah | Synthesis, biological efficiency evaluation and application of sodium alginate-based polyurethane dispersions using cycloaliphatic isocyanate, as antibacterial textile coating[END_REF] or as a disintegrating agent for tablets. Alginate is also commonly found in cosmetic [START_REF] Adli | Development of biodegradable cosmetic patch using a polylactic acid/phycocyanin-alginate composite[END_REF], pharmaceutical, medical [START_REF] Shi | Studies of the properties of CHG-Loaded alginate fibers for medical application[END_REF] Matrix for encapsulation of volatiles constituents.

Coating films to preserve frozen fish.

Pearls from spherification process. As polysaccharides properties are tightly related to theirs structures, the modification of alginate structure has been widely explored. For that, alginate acid hydrolysis showed a uronic acids release through an elimination reaction that led to molecular weight reduction [START_REF] Haug | The Degradation of Alginates at Different pH Values[END_REF]; J. S. [START_REF] Yang | Research progress on chemical modification of alginate: A review[END_REF]. Chhatbar et al. (2009) studied the effect of microwave-assisted acidic hydrolysis of alginate, and reported an acceleration of this reaction with similar oligo-alginate properties than conventional acidic hydrolysis.

Alginate can also be enzymatically and thermally treated [START_REF] Aida | Depolymerization of sodium alginate under hydrothermal conditions[END_REF][START_REF] Hartmann | Enzymatic modification of alginates with the mannuronan C-5epimerase AlgE4 enhances their solubility at low pH[END_REF][START_REF] Kelishomi | Antioxidant activity of low molecular weight alginate produced by thermal treatment[END_REF] to modify its structure and functional properties through the molecular weight reduction or M/G ratio modification. The enrichment with G units results in a rigid structure, when an enrichment with M units produces a flexible molecular structure [START_REF] Vold | A study of the chain stiffness and extension of alginates, in vitro epimerized alginates, and periodate-oxidized alginates using size-exclusion chromatography combined with light scattering and viscosity detectors[END_REF].

Ultrasounds treatment of alginate at different frequencies induce polymer structure degradation, rearrangements and modifications of its molecular weight. A decrease of M/G ratio has also been reported with changes of hydrophobic interactions making alginate treated by 135 kHz having a stronger stiffness compared to the untreated polymer [START_REF] Feng | Molecular weight distribution, rheological property and structural changes of sodium alginate induced by ultrasound[END_REF][START_REF] Taha | Ultrasonic emulsification: An overview on the preparation of different emulsifiers-stabilized emulsions[END_REF].

In addition to their physicochemical properties, biological activities of oligo-alginate obtained by alginate acidic hydrolysis have been investigated. 2. Spherification process

Gelation methods

Two main gelation methods can be presented: (i) the external gelation, also called diffusion process [START_REF] Bajpai | Ca(II)+Ba(II) ions crosslinked alginate gels prepared by a novel diffusion through dialysis tube (DTDT) approach and preliminary BSA release study[END_REF][START_REF] Rezvanain | Optimization, characterization, and in vitro assessment of alginate-pectin ionic cross-linked hydrogel film for wound dressing applications[END_REF]; and (ii) the internal gelation also called pH-controlled gelation as pH determines gelation kinetics [START_REF] Cao | Egg-box model-based gelation of alginate and pectin : A review[END_REF][START_REF] Hernández-González | Alginate hydrogels for bone tissue engineering, from injectables to bioprinting: A review[END_REF]. These two methods are described below.

External gelation

The external gelation method leads to gel formation through diffusion of calcium ions in an alginate solution (X. D.

Internal gelation

Internal gelation method is based on calcium release simultaneously into the all system under controlled conditions. Usually calcium lactate is dissociated to initiate alginate gelation [START_REF] Strobel | Stability of Fish Oil in Calcium Alginate Microcapsules Cross-Linked by In Situ Internal Gelation During Spray Drying[END_REF]. Internal gelation allows a better control of gelation kinetics and produces a homogeneous gel structure with pores. It is used for controlled delivery of active compounds (Hernández-González et al., 2020). As an example, Strobel et al. (2020) reported a significative enhance of fish oil stability after its encapsulation with internal gelation method.

Javvaji et al. (2011) reported a novel light activated gelation method induced by photoacid generation in an insoluble calcium salt dispersion. After 10min of UV-treatment, the released H + ions from the photoacid generator react with the calcium salt and release calcium ions that cross linked with alginate to form a hydrogel.

Spherification process using alginate

Encapsulation process permits to coat a substance for its preservation and/or addition of functional properties. Several encapsulation methods involving alginate can give rise to particles or capsules with several structural organizations. Thus, many parameters have to be considered to choose the appropriate particle structure associated to an encapsulation method: particle size, composition of encapsulated substance, stability of matrix and delivery features.

Besides, those methods have to be scaled up for industrial production (Ching et The beads shape depends on experimental conditions such as pH, calcium concentration and temperature. However, beads sphericity is also determined by alginate solution viscosity and fall distance between nozzle outlet and bath surface. The optimized fall distance to obtain spherical beads has been determined in the range 7 to 10 cm knowing that droplets are formed as a result of solution surface tension. This distance is necessary to the liquid to drag the forces exerted by the gelling bath and overcome the impact to form a spherical bead. Also, sphericity can be improved through surface active compounds addition [START_REF] Blandino | Formation of calcium alginate gel capsules: Influence of sodium alginate and CaCl2 concentration on gelation kinetics[END_REF][START_REF] Ching | Alginate gel particles-A review of production techniques and physical properties[END_REF] Simple extrusion process allowed encapsulation of various compounds for delivery systems and to increase their stability, including oils [START_REF] Bannikova | Microencapsulation of fish oil with alginate: In-vitro evaluation and controlled release[END_REF] In order to produce particles of different sizes (from 20µm to 7mm), numerous encapsulation processes have been derived from simple dripping process. Those methods differ generally by the ways of formation and separation of alginate droplets. Usually, these methods are divided into three groups: jet dispersion, spinning disk and atomization.

• Jet dispersion

The extruded polymer gel is split up following different methods. In this group, vibrating nozzle [START_REF] Pedrali | Encapsulation of grape seed phenolics from winemaking byproducts in hydrogel microbeads -Impact of food matrix and processing on the inhibitory activity towards αglucosidase[END_REF], electrostatic atomization [START_REF] Nikoo | Electrospray-assisted encapsulation of caffeine in alginate microhydrogels[END_REF] and jet cutting (Paulo et al., 2017) are commonly used. It is also possible to merge particular processes for increased efficiency, as the vibrating electrostatic method for example [START_REF] Pedrali | Encapsulation of grape seed phenolics from winemaking byproducts in hydrogel microbeads -Impact of food matrix and processing on the inhibitory activity towards αglucosidase[END_REF].

• Spinning disk

Under centrifugal force, the alginate solution flow forms droplets due to the rotation of the nozzle (or rotating disc). Spinning disk method is used to encapsulate flavor ingredients [START_REF] Yin | Spray-chilling encapsulation of 2-acetyl-1-pyrroline zinc chloride complex using hydrophobic materials: Feasibility and characterization of microcapsules[END_REF]).

• Atomization

Another extrusion method is atomization. The process basis is the extrusion of pressurized polymer solution through a nozzle while injecting pressurized air, atomized particles fall then into a gelling bath. Atomization can be applied for lipid encapsulation for example [START_REF] Strasdat | Incorporation of lipid nanoparticles into calcium alginate beads and characterization of the encapsulated particles by differential scanning calorimetry[END_REF]).

Coextrusion

Coextrusion, also called annular jet method, is an encapsulation technique which allows to obtain core-shell capsules as shown in Fig. 4A. Alginate solution and solutions containing the substances to encapsulate are simultaneously injected through a coaxial nozzle, which permits to obtain an annular jet formed by two concentric jets. The outer material represents the shell material, i.e. alginate solution, and the inner contains the solution with the target substance.

The jet is then separated into droplets and collected in a gelling bath. As for the simple dripping process, different separative methods are possible: vibration, electrostatic forces, spinning disk or simple dropping into crosslinker solution (Oxley, 2012a).

In addition to the previously mentioned factors that impact beads size, a couple of factors are specific to coextrusion process to reach a given shape of core-shell capsules. Coextrusion technology allows production of capsules with a huge payload (over 90%) and a large range of capsules size can be obtained with a diameter from few microns to a several millimeters. The process is easily scaled-up for industrial use (Oxley, 2012a;[START_REF] Wang | Co-extrusion encapsulation of canola oil with alginate: Effect of quercetin addition to oil core and pectin addition to alginate shell on oil stability[END_REF]. For example, Lucia et al (2017) worked on the production of almonds essential oil liquid-core capsules obtained by coextrusion process [START_REF] Lucía | Encapsulation of Almond Essential Oil by Co-Extrusion/Gelling Using Chitosan as Wall Material[END_REF]. [START_REF] Silva | Comparison of extrusion and co-extrusion encapsulation techniques to protect Lactobacillus acidophilus LA3 in simulated gastrointestinal fluids[END_REF] studied the encapsulation of Lactobacillus acidophilus and compared the simple dripping method to the coextrusion technique. An additional protective function was observed for beads produced by coextrusion and especially for capsules obtained with a composite shell made of an alginate/shellac matrix [START_REF] Silva | Comparison of extrusion and co-extrusion encapsulation techniques to protect Lactobacillus acidophilus LA3 in simulated gastrointestinal fluids[END_REF].

Factors influencing alginate gelation and spherification

Molecular weight of alginate

Generally, high molecular weight of alginate due to long polymeric chains results in a larger number of Ca-binding sites then tighter crosslinking in the gel [START_REF] Hu | Characterization of the structure and diffusion behavior of calcium alginate gel beads[END_REF]. These interactions impact gelation process and produce a high viscosity gels, so that low molecular weight alginate weight alginate (1,43x10 5 g mol -1 ) viscosity is 2.4 times lower than higher molecular weight alginate (3,5x10 5 g mol -1 ) viscosity (0.05 Pa.s versus 0.12 Pa.s) in the same experimental conditions.

Gelation kinetics are also faster with increasing alginate molecular weight (Fernández Farrés & Norton, 2014). Moreover, similar conclusions have been found after acidic treatment of high molecular weight alginate so that low molecular weight alginate obtained produced low viscosity gels [START_REF] Haug | The Degradation of Alginates at Different pH Values[END_REF][START_REF] Jiao | Effects of Molecular Weight and Guluronic Acid/Mannuronic Acid Ratio on the Rheological Behavior and Stabilizing Property of Sodium Alginate[END_REF].

Effects of other novel processing methods have been studied. Such as high-pressure homogenization also called dynamic high pressure that caused a reduction of alginate molecular weight and viscosity under 300 MPa and after 5 cycles when a minimal viscosity plateau was observed (Harte & Venegas, 2009).

M/ G ratio and distribution of G, M and GM units along the alginate chains

Alginate gelation is dependent of the percentage of G and M units, as a consequence of a higher degree of hindered rotation around the glycosidic linking. Because of this configuration, cross linking between alginate Ca 2+ is mostly due to G units [START_REF] Braccini | Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: A molecular modeling investigation[END_REF][START_REF] Feng | Molecular weight distribution, rheological property and structural changes of sodium alginate induced by ultrasound[END_REF][START_REF] Jiao | Effects of Molecular Weight and Guluronic Acid/Mannuronic Acid Ratio on the Rheological Behavior and Stabilizing Property of Sodium Alginate[END_REF]. M units moderate elasticity and lateral association in alginate gelation. Gels formed with high percentage of M units are reported as soft and elastic while those formed with high percentage of G units are described as compact and rigid [START_REF] Leirvåg | Strategies for Stabilising Calcium Alginate Gel Beads: Studies of Chitosan Oligomers, Alginate Molecular Weight and Concentration[END_REF]. Beads produced with low molecular weight and high G content alginate are the strongest, the biggest and best relevant to protect probiotics by encapsulation [START_REF] Ramos | Effect of alginate molecular weight and M/G ratio in beads properties foreseeing the protection of probiotics[END_REF].

Alginate Concentration

Gelation can be impacted by alginate concentration. Fernández Farrés et al. ( 2013) found that higher concentration of the biopolymer, in the range of 1% to 4% (w/w), induced higher viscosity, elasticity rupture strength and gel hardness. As expected, higher concentration of alginate molecules increased the number of cross-linking interactions with calcium ions and reinforced gel structure [START_REF] Fernández Farrés | Rheology and tribological properties of Ca-alginate fluid gels produced by diffusion-controlled method[END_REF]. However, excessive concentration of alginate reduced binding opportunities by decreasing Ca 2+ diffusion through the matrix (J. [START_REF] Yang | Gelation kinetics of alginate chains through covalent bonds[END_REF]. Nevertheless, for moderate changes in alginate concentration from 1.3 to 1.7% [w/w], Hu et al. ( 2020) did not notice significant variations of alginate beads structure.

In the case of beads processing, alginate concentration has also an impact on beads shape in alginate extrusion process, alginate beads tend to be more spherical when viscosity increases.

The result of a bead made with the appropriate viscosity can be found in Fig. 3C.

Nevertheless, when viscosity is too high, beads are drop-shaped as shown in Fig. 6

Concentration of calcium ions

An increase of Ca 2+ concentration results in an increase of cross-linking gelation rate. Faster diffusion of calcium ions in gels lead to higher gel viscosity, elasticity, and stability (J. Higher viscosity and maximum tensile stress of alginate gel are reached at around 1.4% (w/w) of [CaCl2] for alginate concentration equaled to 127mM. Ca ++ saturation of alginate chains can explain that maximum tensile stress occurred [START_REF] Cuadros | Mechanical properties of calcium alginate fibers produced with a microfluidic device[END_REF]. At high calcium concentration calcium ions induce excessive positive changes that will segregate alginate chains leading to the gel disintegration. The mechanism between alginate structure and calcium chloride concentration is illustrated in Fig. 7.

It should be noted that, while producing alginate beads, a high calcium concentration also affects beads sphericity which tends to be less spherical at high calcium concentrations [START_REF] Cuadros | Mechanical properties of calcium alginate fibers produced with a microfluidic device[END_REF].

Residence Time

The time during which alginate is into contact with calcium ions in gelation bath is called residence time. When alginate drops are exposed to calcium, ions migrate from surface to core. Required residence time for reticulation (tgel) can be calculated using Fick's second law which can be expressed as:

∂t = ∂ C ∂
Where C is alginate concentration (mol/m 3 ), t is time (s), D is diffusion substance coefficient (m 2 /s) and x is the position (m).

Fick's second law can be approximated as:

≈ C
Where C represents alginate concentration (mol/m 3 ), L is a characteristic length given by the bead radius (m), and D the diffusion coefficient of calcium chloride in water: 10

Ionic strength 298

Gelation is impacted by solution ionic strength. Low ionic strength allows gel formation, but 299 high level of monovalent salts competed with calcium ions for carboxyl alginate groups 300 resulting in an instable and weak system. Van Leusden et al. (2017) reported a structural 301 alteration of alginate beads exposed to high NaCl concentration. This modification might be 302 due to a decrease of electrostatic attractive interactions into the system and/or increase in 303 beads pore size resulting in the release of the encapsulated compounds [START_REF] Jiao | Effects of Molecular Weight and Guluronic Acid/Mannuronic Acid Ratio on the Rheological Behavior and Stabilizing Property of Sodium Alginate[END_REF]. 304

Apparently, salts ions dislocated some Ca 2+ leading to gel disintegration. The extent of 305 network destructuration increased with higher salts concentration [START_REF] Jiao | Effects of Molecular Weight and Guluronic Acid/Mannuronic Acid Ratio on the Rheological Behavior and Stabilizing Property of Sodium Alginate[END_REF]. 306 3.9. Shear rate 307 [START_REF] Jiao | Effects of Molecular Weight and Guluronic Acid/Mannuronic Acid Ratio on the Rheological Behavior and Stabilizing Property of Sodium Alginate[END_REF] reported the influence of shear rate on the gelation method described as "in 308 situ addition of calcium", to favor homogenous gelation by avoiding disadvantages of 309 external gelation. Shear rate had an impact on gelation kinetics: when shear was applied at the 310 beginning of gelation process, the onset time of gelation was higher and gel particles were 311 faster formed (Hernández-González et al., 2020; Jiao et al., 2019; [START_REF] Puguan | Characterization of structure, physico-chemical properties and diffusion behavior of Ca-Alginate gel beads prepared by different gelation methods[END_REF]. 312

However, gel viscosity decreased with increasing shear rate applied demonstrating the 313 relation between shear rate and gel rheology [START_REF] Mancini | Fruit-alginate interactions in novel restructured products[END_REF]. 314

315

In conclusion for factors influencing alginate gelation and spherification, Table 2 summarizes 316 the effect of the principal parameters on alginate beads gelation. We choose to present the 317 clearer effect for an efficient production of alginate bead with desired properties. Some 318 parameters have not been enough studied to appear in our summarized Table 2

↗ ↗ ↘ ↘ ↗ ↗↘ ↗ ↗M/G ratio ↘ ↘ ↘ - - ↘ - ↗ Experimental environment ↗ [alginate] ↗ ↗ ↗ ↘ ↗ ↗ ↘ ↗ [Ca 2+ ] ↗ ↗ - ↗ - - ↘ ↗Temperature Optimum at 36-40°C ↘ ↘ ↗ ↘ - ↗ ↘

Composite Alginate gels

Composite gels with alginate and other polymers have been studied for multiple purposes and in many fields, such as encapsulation, packaging films, or edible films. This interest results from the ambition to modify gel mechanical properties [START_REF] Poncelet | Production of alginate beads by emulsification/internal gelation[END_REF], brings new characteristics and extends its use. Polysaccharides and proteins are the main components used for copolymerization. Depending on copolymer nature mixed with alginate, gelation process varies as described below.

Gum arabic

Also known as acacia gum, gum arabic is a natural polymer extracted from acacia trees and it consists of β-D-galactopyranosyl units branched with side chains of L-rhamnose, D-galactose, L-arabinose and D-glucuronic acid. This polymer is largely used in food, cosmetic and pharmaceutical industries as thickening agent and stabilizer [START_REF] Tsai | Effect of gum arabic-modified alginate on physicochemical properties, release kinetics, and storage stability of liquid-core hydrogel beads[END_REF]. Interaction between alginate and gum arabic has been recently studied by [START_REF] Sabet | The interactions between the two negatively charged polysaccharides: Gum Arabic and alginate[END_REF]. These authors reported interactions between polymers at pH 7 and a stronger synergy at pH 4, due to ionic interactions between respective negative and positive charges of alginate and gum arabic. The ionic binding between alginate and gum arabic results in an increase of gel viscosity and viscoelasticity (Sabet et

Chitosan

Chitosan is a linear polysaccharide extracted from crustaceans' shells chitin. It is a nontoxic biopolymer formed by D-glucosamine and N-acetyl glucosamine units. Chitosan is used in numerous field due to its numerous properties, i.e. antioxidant, lipid binding, antiinflammatory and antimicrobial effects [START_REF] Kulig | Study on alginate-chitosan complex formed with different polymers ratio[END_REF].

Chitosan gel viscosity increases with pH. The strongest alginate-chitosan gels were obtained with increasing pH and chitosan weight percentage, but considerable viscoelasticity and maximum encapsulation capability were obtained at neutral pH [START_REF] Afzal | Rheological behavior of pH responsive composite hydrogels of chitosan and alginate: Characterization and its use in encapsulation of citral[END_REF].

Combining alginate and chitosan improves mechanical and chemical gel properties and favors thermal resistance. Chitosan has been largely investigated in coating alginate capsules, due to its positive charges that lead to an ionic complexation with negative charges of alginate. This complexation allows chitosan solubilization into a non-acetic acid or hydrochloric solutions.

Thereby, coating with chitosan enhanced capsules membrane thickness and their protective abilities [START_REF] Fernandes | Physicochemical properties and microbial control of chestnuts (Castanea sativa) coated with whey protein isolate, chitosan and alginate during storage[END_REF][START_REF] Raeisi | Effects of Sodium Alginate and Chitosan Coating Combined with Three Different Essential Oils on Microbial and Chemical Attributes of Rainbow Trout Fillets[END_REF][START_REF] Taqieddin | Enzyme immobilization in novel alginate-chitosan core-shell microcapsules[END_REF].

Composites exhibited great results for globulin preservation during thermal treatment [START_REF] Kulig | Study on alginate-chitosan complex formed with different polymers ratio[END_REF][START_REF] Romo | Soluble complexes between chenopodins and alginate/chitosan: Intermolecular interactions and structural-physicochemical properties[END_REF]. De Prisco et al (2015) reported a better stress tolerance to food processing for probiotics encapsulated with alginate-chitosan composites.

Alginate/chitosan composites have been examined for oil co-encapsulation or microencapsulation to protect oil from oxidation, capsules showed good oxidative stability and oxidation products were lower for chitosan/alginate compared to non-encapsulated oil.

Results also showed an effective oil vehicle for nutraceuticals [START_REF] Atencio | Encapsulation of ginger oil in alginate-based shell materials[END_REF][START_REF] Hamed | Edible alginate/chitosan-based nanocomposite microspheres as delivery vehicles of omega-3 rich oils[END_REF]. Microwave treatments have also been considered for drugs controlled-release alginate/chitosan microbeads. Depending on experimental conditions, microwave treatments could induce cross-linkage or breakdown. Thus, [START_REF] Wong | Design of controlled-release solid dosage forms of alginate and chitosan using microwave[END_REF] reported a retarded drugs release from alginate/chitosan beads treated at 80 W for 10 minutes as a consequence of enhanced alginate/chitosan cross-linking.

Carrageenan

Carrageenan is a sulphated polygalactan that can be obtain from red seaweeds, it contains 15 to 54% charged sulphate ester groups. This polysaccharide is formed by D-galactose and 3,6anhydro-galactose linked by α-1,3 and β-1, 4-glycosidic bonds. There are various carrageenan types: λ, κ, ι, ε, μ. The classification depends on the sulphate ester groups number and position [START_REF] Necas | Carrageenan: A review[END_REF]. Alginate/carrageen composite have been largely investigated in edible films formulation for food and non-food applications; good barrier and protection properties have been reported (Tavassoli-Kafrani et al., 2016).

Combination of κ-carrageenan and alginate enhances gel rigidity and mechanical stability.

Carrageenan/alginate composite gel appeared more transparent than conventional alginate gel with a compact and smooth surface. Addition of carrageenan tends also to improve thermal properties, elastic modulus, gel water content, and swelling which is induced by sulphate groups from carrageenan molecules. Beads formed with carrageenan/alginate composite had a larger diameter than alginate beads (L. Li et al., 2019;[START_REF] Mohamadnia | Ionically cross-linked carrageenan-alginate hydrogel beads[END_REF].

The pH-dependent properties allowed use of carrageenan/alginate complex in a pHresponsive beads for insulin encapsulation. In this study, protection against acidic condition and controlled release were investigated [START_REF] Lim | Controlled delivery of oral insulin aspart using pHresponsive alginate/κ-carrageenan composite hydrogel beads[END_REF]. Good protection was observed at pH=1.2 and progressive release with good kinetics was noticed at pH=7.4. This point made composite carrageenan/alginate beads promising encapsulation matrix for oral insulin intake. alginate/whey protein isolate composite for probiotics encapsulation and reported a highest survivability of lactobacillus acidophilus for an optimal matrix composed of 4.54% (w/v) of alginate and 10% (w/v) of whey protein isolate [START_REF] Dehkordi | Optimization of Alginate-Whey Protein Isolate Microcapsules for Survivability and Release Behavior of Probiotic Bacteria[END_REF].

Recent studies showcased interest of protein addition in edible alginate films. Chkravartula et al (2019) reported significant beneficial effects of whey protein on the physico-chemical, rheological, mechanical and optical properties of edible packaging. Addition of whey proteins to alginate decreased film viscosity and enhanced its hydrophobicity and reduced film water absorption [START_REF] Chakravartula | Characterization of composite edible films based on pectin/alginate/whey protein concentrate[END_REF].

Gelatin

Gelatin is largely studied in coacervation process, which is a spontaneous phase separation between an insoluble complex and of another aqueous phase that occurs with pH change (de

5. Other Encapsulation processes using alginate

Spray-drying

Spray-drying is known since the 1930's as one of the earliest encapsulation techniques due to its simplicity of application and high productivity among many other advantages (Table 4) (Z.

Fang & Bhandari, 2012). The process principle is injection of a solution (emulsion or dispersion with active compounds) through a simple nozzle or centrifugal wheel. The liquid is then atomized into small droplets which fall and meet hot air in a drying chamber. After water evaporation, particles fall to the bottom of the chamber and are collected in a powder form.

Spray-drying process is applied in many fields and several wall materials can be used.

Alginates exhibit good results for volatiles and emulsions encapsulation (Z. [START_REF] Fang | Spray drying, freeze drying and related processes for food ingredient and nutraceutical encapsulation[END_REF][START_REF] Rodríguez | Current encapsulation strategies for bioactive oils: From alimentary to pharmaceutical perspectives[END_REF].

The efficiency of spray-drying process depends strongly on inlet air temperature, particularly for thermolabile substance encapsulation, and can affect particles chemical and physical properties in addition to core materials stability (X. [START_REF] Sun | Effect of spray-drying temperature on physicochemical, antioxidant and antimicrobial properties of pectin/sodium alginate microencapsulated carvacrol[END_REF]. In this perspective, X.

Sun et al (2020) studied the impact of inlet air temperature on spray-drying microencapsulation of lipophilic carvacrol in an oil-in-water emulsion with alginate/pectin composite shell material. They reported an optimum temperature for spray-drying encapsulation between 100 and 130°C for carvacrol. However, even if a higher temperature tends to increase encapsulation efficiency, it leads to reduce moisture content and retention efficiency (X. Stobel et al (2020) worked on fish oil encapsulation using the CLAMs process and reported good results in term of shelf-life of encapsulated fish oil in a shell material made of modified starch and alginate. Some promising enteric release properties were also observed for gastrointestinal delivery of lipophilic bioactives [START_REF] Strobel | Stability of Fish Oil in Calcium Alginate Microcapsules Cross-Linked by In Situ Internal Gelation During Spray Drying[END_REF].

Spray-cooling

Also known as spray-congealing or prilling, spray-cooling is a process that allows atomization of a liquid into small particles. The process is similar to spray-drying, the solution is injected through a pressure nozzle, vibrating nozzle or a spinning disk into an atomization chamber.

Unlike spray-drying, the atomized liquid is cooled and hardened [START_REF] Oxley | Spray-cooling and spray-chilling for food ingredient and nutraceutical encapsulation[END_REF].

Encapsulation by spray-cooling allows encapsulation of actives ingredients dispersed into a liquid matrix. Instead of spray-drying, encapsulation by spray-cooling allows encapsulation of thermolabile substances [START_REF] Oxley | Spray-cooling and spray-chilling for food ingredient and nutraceutical encapsulation[END_REF]. This process is used in food and nutraceutical industry for preservation of flavor [START_REF] Oxley | Spray-cooling and spray-chilling for food ingredient and nutraceutical encapsulation[END_REF]

Coacervation

Coacervation occurs in a colloidal system and leads to liquid/liquid phase separation between a colloid-rich phase also known as coacervate phase and a solvent-rich diluted phase. The coacervate phase can be composed of a single polyelectrolyte. In this case, it is called simple coacervation. Complex coacervation occurs when the coacervate phase is composed of an ionic complex of two opposite charges polyelectrolytes as for alginate complex coacervation for encapsulation purposes. Solution pH strongly impacts coacervate formation, in addition to polyelectrolytes concentrations and their mixing ratio, ionic strength and temperature [START_REF] Gorji | Complex Coacervation of Milk Proteins with Sodium Alginate[END_REF][START_REF] Thies | Microencapsulation methods based on biopolymer phase separation and gelation phenomena in aqueous media[END_REF].

Combination of proteins and alginate is often studied for delivery systems, and in many fields such as food, cosmetic and pharmaceutical industries. For olive oil encapsulation, microcapsules sizes increase with the increase of oil amount but decreases with the increase of surfactant amount. Besides, FTIR spectra of free olive oil and microencapsulated oil have shown same characteristics so that no interactions between oil and alginate/gelatin composite have been exhibited [START_REF] Devi | Study of complex coacervation of gelatin a and sodium alginate for microencapsulation of olive oil[END_REF].

Conclusion

Alginate is a widely used and investigated polymer for a large variety of technical applications as thickener or stabilizer but also in spherification process for its gelation ability to encapsulate sensible materials. It is Generally Recognized as Safe (GRAS), being nontoxic, nonantigenic, satisfactorily biocompatible, and sufficiently biodegradable.

Alginate gelation depends on polymer chemical structure counting its molecular weight and G/M units ratio and distribution along the alginate chain. Experimental environment greatly influences alginate gelation including pH, temperature, polymer concentration, crosslinker concentration, residence time and ionic strength.

Alginate has the advantage to be an encapsulation material adapted for most encapsulation process especially spherification (extrusion and coextrusion) through its biocompatibility and capability to form a hydrogel through ionic cross-links with divalent cations, such as calcium ions. However, alginate has some limitations in certain applications. Therefore, alginate can work in synergy with many polymers (e.g. polysaccharides and proteins). Thus, composite alginate gels are is widely studied recently in order to improve alginate encapsulation and spherification properties, including encapsulation efficiency and payload, storage stability and barrier properties.

Alginate is certainly one of the most promising encapsulation materials.

1. 1 .

 1 Alginate molecular structure and its modification by processing methods Alginate is formed by a chain of β-D-mannuronic acid (M units) and α-L-guluronic acid (G units), linked via (1→4) glycosidic linkage. The percentage of those units varies among algae species (Rhein-Knudsen et al., 2017) and impacts alginate properties (Hecht & Srebnik, 2016).

Fig. 1 .

 1 Fig. 1. Structural characteristics of alginate. M: mannuronate residue; G: guluronate residue.

  calcium ions replace sodium to cross-link two alginate chains and form a network (Vinet & Zhedanov, 2011; Warsinger et al., 2015). Alginate gelation is commonly described as an eggbox model formed by two antiparallel chains of polyuronates linked with Ca 2+ binding blocks as illustrated in fig.2 (Martău et al., 2019; Reynolds & Enquist, 2006). Stokke et al. (2000) suggested a lateral association of G units in the eggbox structure.

Fig. 2 .

 2 Fig. 2. Alginate gel structure in presence of calcium ions (egg-box model) (Adapted from Martău et al., 2019)

  and trap it into the matrix[START_REF] Ching | Alginate gel particles-A review of production techniques and physical properties[END_REF]. Different structural organization can be produced depending on the extruded solution. For instance, an alginate solution loaded with the target compound tends to produce beads with homogenous microstructure Fig.3A. An emulsion consisting in a lipidic phase dispersed in an aqueous phase of alginate results in obtention of beads with a dispersed microstructure Fig.3B.

Fig. 3 .

 3 Fig. 3. Scheme of homogeneous structural organization for capsules (A), scheme of dispersed structural organization for capsules (B) and a photograph of a homogenous capsule.

  For instance, nozzles sizes (internal/external) affect capsules size. Slow flow rate tends to produce larger capsules (drop by drop system) than capsules produced at high flow rate (Lucía et al., 2017; Oxley, 2012a).

Fig. 4 .

 4 Fig. 4. Scheme of a core-shell structural organization for capsules (A), scheme of a combination structural organization for capsules (B) and a photograph of a core-shell capsule.

Fig. 5 .

 5 Fig. 5. Effect of calcium on the MG, MM and GG alginate units.

  (Z. Q. Li et al., 2013).

Fig. 6 .

 6 Fig. 6. Example of a drop-shaped alginate bead.

  [START_REF] Li | A new insight to the effect of calcium concentration on gelation process and physical properties of alginate films[END_REF]. The calcium versus carboxyl group concentrations ratio is called the R value and describes effect of Ca 2+ on alginate gelation and it is defined as follows:R = [Ca 2+ ]/[COO-]Alginate crosslinking with calcium ions can be described in three steps: (i) the monocomplexation at R < 0.25, (ii) dimerization at 0.25<R<0.55 resulting in formation of the eggbox model; and (iii) third step occurs at R > 0.55 and results in the lateral association of the polymer chains forming intra of inter-clusters (Y.[START_REF] Fang | Multiple Steps and Critical Behaviors of the Binding of Calcium to Alginate[END_REF].

Fig. 7 .

 7 Fig. 7. Scheme representing the mechanism of strengthening and weakening of the alginate gel as a function of the concentration of calcium chloride (Adapted from Cuadros et al., 2012)

Fig. 8 .

 8 Fig. 8. Macrostructure of alginate beads synthetized at pH= 3.8 and pH ≥ 5.3.7. TemperatureGelation temperature has an impact on alginate gel microstructure. High temperature seems to enhance and facilitate calcium ions penetration through alginate network while low temperature slows down diffusion and gelation process. Low temperature led to more ordered and regular network and higher temperature produced a denser network with lower young's modulus[START_REF] Jeong | Changes in the physical properties of calcium alginate gel beads under a wide range of gelation temperature conditions[END_REF]. At physiological gelation temperature around 36-40°C, an alginate gel with high young's modulus was produced[START_REF] Leo | Effects of Sterilization Treatments on Some Properties of Alginate Solutions and Gels[END_REF][START_REF] Oates | Studies on the effect of heat on alginates[END_REF]).

4. 1 .

 1 Polysaccharides as co-polymer 4.1.1. Pectin Pectin is a biopolymer extracted from plant cell walls like citrus peel or apple pomace. B. Zhang et al. (2019) reported that pectin gelation is enhanced by addition of guluronic units. Encapsulation in alginate-pectin gel exhibited a significant reduction of anthocyanins degradation (Guo et al., 2018; B. Zhang et al., 2019). A lower energy consumption has also been demonstrated in jelly candy manufacturing using alginate-pectin cold gelation instead of traditional heating process (de Avelar & Efraim, 2020). In the medical field, alginate-pectin hydrogel presented great improvement of mechanical properties, water absorption, thermal stability, and drug release for wound dressing application (Oh et al., 2020; Rezvanain et al., 2017). In food industry, pectin-alginate-gelatin composite gels showed promising results for probiotics encapsulation, by increasing stability of encapsulated bacteria (Vaziri et al., 2018). 4.1.2. Gums 4.1.2.1. Guar gum Guar gum is extracted from guar beans (Cyamopsis tetragonoloba). It is composed of a linear chain of (1-4)-β-D-mannopyranose linked with β-galactopyranosyl groups. Guar gum is water soluble and usually used in coating. Guar gum is used as thickener of alginate-guar gum gel (Mousavi et al., 2019; Rastegar & Atrash, 2020). Gel made with alginate/guar gum was stronger at lower calcium level that pure alginate gel and presented a high elasticity and rigidity due to a lower degree of porosity. It reduced syneresis and shrinkage during storage. Guar gum addition to alginate enhanced network stability in acidic conditions and thermal processing (Mousavi et al., 2019, 2020). Alginateguar gum gel exhibited an improved polyphenols and betacyanins encapsulation (Aguirre Calvo et al., 2019), and enhanced preservation of antioxidant activity for encapsulated fatty acids (Aguirre-Calvo et al., 2020).

  L.[START_REF] Gu | Formulation of alginate/carrageenan microgels to encapsulate, protect and release immunoglobulins: Egg Yolk IgY[END_REF] worked on immunoglobulin encapsulation for controlled release and reported high encapsulation efficiency. Composite exhibited a denser internal microstructure and a good protection in acidic gastric conditions (L.[START_REF] Gu | Formulation of alginate/carrageenan microgels to encapsulate, protect and release immunoglobulins: Egg Yolk IgY[END_REF]. 4.1.5. Starch One of the most abundant and cheap natural polysaccharides is starch. Starch contains amylose and amylopectin with linkage of α-D-glucopyranose molecules. Starch is largely used in hydrogel production, due to its free hydroxyl groups. Starch works in synergy with alginate. Water absorption capacity and encapsulation efficiency are improved. For instance, López Córdoba et al. (2013) reported improvement of encapsulation efficiency from 55 to 65% of liquid extract of yerba mate (a high polyphenols tea-like beverage) (da Silva Fernandes et al., 2019; López Córdoba et al., 2013). The synergy between starch and alginate depends on chemical structures of both polymers, but also on processing conditions, such as pH, ionic strength and salts concentration (da Silva Fernandes et al., 2019). Gelation of alginate/starch composite occurs using different divalent cations (Mn 2+ , Zn 2+ , Ca 2+ for example), and characteristics of the composite gel vary from an ionic crosslinker to another (da Silva Fernandes et al., 2019). Addition of starch to alginate creates composite gels with variable porosity depending on starch type, but starch addition provides a slower release of encapsulated substances (Feltre, Almeida, et al., 2020; Okunlola & Adewusi, 2019). Starch type impacts gel water retention, stability, and digestibility. Those features must be considered in the formulation of controlled release beads. For instance, Hassan et al (2020) obtained promising results for gradual release of nisin encapsulation (Feltre, Almeida, et al., 2020; Hassan et al., 2020). Feltre, Sartori, et al (2019) recently reported an efficient encapsulation of oil with starch incorporation in alginate systems, due to greater oil dispersion into oil/starch/alginate emulsion. Beads formed by oil/starch/alginate emulsions containing equal proportions of alginate and starch presented good results in terms of antioxidant activity preservation and storage stability (Feltre, Sartori, et al., 2020). Similar results concerning encapsulated component stability have been obtained by Jain et al (2020) for lycopene encapsulation with an enhanced bioaccessibility. This could be promising for nutraceuticals and functional foods application (Jain et al., 2020). As an example, alginate/starch/gelatin composite has been recently investigated for encapsulation of β-carotene in oil/starch/alginate/gelatin emulsion.The study demonstrated a great stability and oxidation reduction for encapsulated β-carotene compared to β-carotene in free oil. Those results are very important to extend shelf-life of components sensitive to oxidation during processing for food or drugs application (Guedes[START_REF] Silva | Protection and targeted delivery of β-carotene by starch-alginate-gelatin emulsion-filled hydrogels[END_REF].

4. 2 .

 2 Proteins/ polypeptides as co-polymer 4.2.1. Whey Proteins Under specific conditions, proteins can be used as cations source for alginate gel crosslinking. Thus, alginate/proteins composite hydrogels are largely investigated and exhibited great results in protein carriers with controlled release kinetics (Rahmani & Sheardown, 2018). Denatured whey protein isolates and alginate composite gels have shown great thermal stability after 1 minute at 63 and 72°C and a good resistance to acidic conditions for encapsulated probiotics by extrusion or spray-drying process (H. Liu et al., 2018; Praepanitchai et al., 2019; Rajam et al., 2012). Dehkordi et al (2020) worked on an

  Matos et al., 2018). Consequently, gelatin and alginate are pertinent materials for encapsulation process by coacervation. Promising results have been obtained for essential oils microencapsulation, especially with a 4% gelatin and high essential oils concentration matrix, with a 73.7% encapsulation efficiency, and a good terpenes preservation (de Matos et al., 2018; Heckert Bastos et al., 2020). Moreover, Y. Li et al (2019) reported a good effectiveness of alginate/arabic gum /gelatin composite for preservation of encapsulated antioxidant substances for oral delivery (Y. Li et al., 2019). Lemos et al (2017) also observed high encapsulation efficiency in the production of buriti oil microcapsules through coacervation (Lemos et al., 2017).M. Lopes et al (2017) worked on probiotics encapsulation using an alginate/gelatin composite matrix. Spherical and regular beads were obtained, and higher cells viability was reported at

  and food industries, as thickener and stabilizer (Senturk Parreidt et al., 2018; C. Su et al., 2018; H. Zhang et al., 2019).

	Table 1
	Alginates types and their applications
	Code	Alginate types	Applications	References
	E400	Alginic acid	Zero calorie ingredients for diet foods	Mancini & McHugh, 2013.
			(biscuits, low-sugar marmalade, ice-
			creams).
	E401	Sodium alginate	Stabilizer and thickener in frostings,
			puddings and processed fruits and fruit
			juices.

  major use of alginate today is the encapsulation technology through its biocompatibility and capability to form a hydrogel through ionic cross-links with divalent cations, such as calcium ions (B. B.[START_REF] Lee | Air Extrusion System for Ionotropic Alginate Microgel Particle Formation: A Review[END_REF][START_REF] Martău | The Use of Chitosan , Alginate , and Pectin in the Biomedical and Food Sector _ Biocompatibility, Bioadhesiveness, and Biodegradability[END_REF][START_REF] Ramos | Effect of alginate molecular weight and M/G ratio in beads properties foreseeing the protection of probiotics[END_REF].

				Norton et al., 2010; Qin et
	E402	Potassium alginate		al., 2018.
	E403	Ammonium alginate
	E404	Calcium alginate
	E405	Propane-1,2 diol	Stabilizer of emulsion in acidic environment	Norton et al., 2010.
		alginate	(salad dressing).
			Stabilizer of foam in brewing industry.

A

  Plant growth bio-stimulating activity was demonstrated(Salachna, at al., 2018). Anti-allergic activity of a 13.3 kDa oligo-alginate was reported after scission of an initial 1 605 kDa alginate polymer by a 3kGy alpha-One of the most studied properties of alginate, is its ability to form hydrogels by crosslinking with divalent or trivalent cations such as Ca 2+ , Cu 2+ , Sr 2+ , Al 3+ , for example. Those cations present various affinities toward alginate, that result in hydrogels with different structural and mechanical characteristics (Young modulus, gel morphology, etc)(Agulhon et

	irradiation (Eu, et al 2008).
	1.2. Ion-binding for alginate gelation

al., 2012; Kabir et al., 2020; Vinet & Zhedanov, 2011).

For food or cosmetic application, Ca 2+ is the most commonly used. So, in the literature, calcium alginate gels are the most largely investigated. Gelation occurs in solution when

  [START_REF] Liu | Characterization of structure and diffusion behaviour of Ca-alginate beads prepared with external or internal calcium sources[END_REF]. Alginate solution droplets are usually extruded into a calcium bath; this method is called conventional or classical spherification. A solution

containing calcium ions can also be dropped into an alginate bath and this process is known as inverse spherification.

Gel prepared by external gelation presents an inhomogeneous structure with low mechanical properties, this is due to the presence of calcium ions mostly at the surface

(

Arriola et al., 2019; McClements, 2017; Spadari et al., 2019; Q. Sun et al., 2018). A concentration gradient of polymer/ions exists from beads surface to core (Sagdicoglu Celep et al., 2020). Several researches focused on this feature to overcome it (Hernández-González et al., 2020; Puguan et al., 2014), or to study bioactive substances delivery systems in food and medicine applications (X. D. Liu et al., 2002; Mancini & McHugh, 2000).

  al., 2017).

	2.2.1. Extrusion
	2.2.1.1. Simple dripping
	Simple extrusion process is the most commonly spread encapsulation way. The principle
	consists in extrusion through a nozzle of an alginate solution or an emulsion containing
	alginate. The formed droplets fall into a gelation bath containing an alginate crosslinker
	(generally Ca 2+ ) under stirring. Following this basic principle, the process can be varied to
	incorporate interesting components into alginate solution/emulsion in order to encapsulate it

  (2012). Residence time seems to have no effect on beads sphericity[START_REF] Hartmann | Enzymatic modification of alginates with the mannuronan C-5epimerase AlgE4 enhances their solubility at low pH[END_REF].

	3.6. pH
	pH impacts alginate water solubility and ion-binding as it determines the electrostatic charges
	of G and M units. M and G units have pKa of 3.38 and 3.65, respectively. Alginate polymer
	residues have pKa value usually estimated around 3.4 (Park et al., 2019). Alginate composed
	of a high content of MG-sequences are more soluble at low pH and precipitate at pH around
	2.6 and 2.7 while high G units content alginate precipitate at pH around 3.25 (Haug, Larsen,
	Samuelsson, et al., 1963; Sonego et al., 2016; Zazzali et al., 2019). At higher pH values
	(below pH = 10.5) glycosidic bonds of alginate gel start to weaken and break leading to
	structural degradation (Zazzali et al., 2019).

-9 m 2 /s

[START_REF] Evageliou | Effect of pH, sugar type and thermal annealing on highmethoxy pectin gels[END_REF]

. It has been demonstrated that after a residence time of

[START_REF] De Prisco | Microencapsulation by vibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to enhance its survival in foods and in gastrointestinal environment[END_REF]

min, samples exposed to different calcium concentrations showed similar behaviors (Cuadros et al. The microstructure of the alginate gel has been largely studied and it has been established that gel obtained at pH = 3.8 has a denser and interconnected microstructure due to stronger chains interactions. This phenomenon leads to less syneresis and shrinkage of the gel. Gels produced pH ≥ 5 present lower interconnectivity in their microstructure and lower density, then allowing syneresis and shrinkage (Augst et al., 2006; Jeong et al., 2020; Moe et al., 1992; Topuz et al., 2012).

During preparation of alginate beads, interfacial tension is impacted by pH, lower pH values result in lower interfacial tension. pH also influences beads shape, beads produced in low pH gelation bath tend to be oblate, particularly in low alginate and ions concentrations, while those produced at pH > 4 tend to be spherical

[START_REF] Chuang | Effects of pH on the Shape of Alginate Particles and Its Release Behavior[END_REF][START_REF] Sonego | Ca(II) and Ce(III) homogeneous alginate hydrogels from the parent alginic acid precursor: A structural study[END_REF][START_REF] Zazzali | Effects of pH, extrusion tip size and storage protocol on the structural properties of Ca(II)-alginate beads[END_REF]

.

Table 2 321

 2 Effects of increased factors influencing alginate gelation and spherification. 322

	and factors as

  al., 2021). The alginate/gum arabic composite exhibited great results in drug encapsulation. Moreover, Nayak et al (2012) reported an encapsulation efficiency of 86.02 ± 2.97%. 4.1.2.3. Other gums Other gums such as galbanum gum or basil seed gum have been investigated recently for different applications, especially in food packaging field. Addition of gum to alginate matrix brings a real advantage in terms of barrier function against gas, water, oil transport (Hamedi et al., 2017; Mousavi et al., 2020).

Table 3

 3 Co-polymers for alginate composite gels for encapsulation.

	Co-polymers	Composition	Positive effect of co-polymer addition to alginate	Application	Refs
	Pectin	α-D-galacturonic acid	Encapsulated substance stability.	Probiotics encapsulation.	Belscak-Cvitanovic et al., 2015;
		α-L-rhamnose	Gel water absorption and thermal stability.	Cold gelation for candy manufacturing.	de Avelar & Efraim, 2020; Oh
			Lower gel viscosity.		et al., 2020; Vaziri et al., 2018
	Guar gum	β-D-mannopyranose	Strong gel	Coating for food packaging	Aguirre-Calvo et al., 2020;
		β-galactopyranosyl	Low porosity gel	Fatty acids encapsulation	Mousavi et al., 2019; Rastegar
			Network stability to heat and acidic conditions		& Atrash, 2020
	Gum arabic	β-D-galactopyranosyl	High viscosity and viscoelasticity gel	Drugs encapsulation	Nayak et al., 2012; Sabet et al.,
		L-rhamnose	Encapsulation efficiency	Radish juice liquid-core encapsulation	2021; Tsai et al., 2017
		D-galactose
	Chitosan	D-glucosamine	Mechanical, chemical properties and thermal stability.	Protein encapsulation	Fernandes et al., 2020; Hamed
		N-acetyl-glucosamine	Gel protective abilities and permeability	Omega-3 rich oil encapsulation	et al., 2020; Kulig et al., 2016;
				Coating capsules	Romo et al., 2020
	Carrageenan	D-galactose	Gel rigidity and mechanical stability (elastic modulus).	Immunoglobulins encapsulation	L. Gu et al., 2021; L. Li et al.,
		3,6-anhydro-galactose	Water content, thermal properties, and swelling.	Edible films for food application	2019; Necas & Bartosikova,
					2013; Tavassoli-Kafrani et al.,
					2016
	Starch	Amylose	Water absorption. Better oxidation stability	Spirulina encapsulation	Feltre, Sartori, et al., 2020; Jain
		Amylopectin	Capsule digestibility	Edible films for food packaging	et al., 2020; Rajmohan &
		α-D-glucopyranose		Nisin or	Bellmer, 2019; Wu et al., 2001
				lycopene encapsulation
	Whey protein	α-lactalbumin	Gel antioxidant properties	Films for food packaging	Chakravartula et al., 2019;
		β-lactoglobulin	Active protection against visible light, heat, and acidic	Probiotics encapsulation	Dehkordi et al., 2020; Oliveira
		Albumin	conditions		Filho et al., 2019; Praepanitchai
		Immunoglobulins			et al., 2019
	Gelatin	Amino acids	Encapsulation efficiency	Essential oils encapsulation	Heckert Bastos et al., 2020; Y.
			Set up coacervation process	Probiotics encapsulation	Li et al., 2019; S. Lopes et al.,
				Antioxidant encapsulation	2017

  It uses a calcium ions source which is insoluble at feed pH, but when solution is atomized the base present in the matrix evaporates which drop its pH and allows calcium ions to solubilize and react with alginate. The advantage of this process is to reduce the multi-step conventional spray-drying process into a one-step operation[START_REF] Jeoh | How alginate properties influence in situ internal gelation in Crosslinked Alginate Microcapsules (CLAMs) formed by spray-drying[END_REF][START_REF] Strobel | Industrially-Scalable Microencapsulation of Plant Beneficial Bacteria in Dry Cross-Linked Alginate Matrix[END_REF].

Sun et al., 2020). Recently, Stobel et al. (2018) presented a new encapsulation process using spray-drying technology and called the Cross-Linked Alginate Microcapsules process (CLAMs process).

  , vitamins (Chalella Mazzocato et al., 2019), or other bioactive ingredients like probiotics (Dong et al., 2013).

  encapsulation using 4% of gelatin and 10% of alginate. Microcapsules with a 434 µm diameter have been obtained with promising results for controlled release (de Matos et al., 2018).

	Beta-lactoglobulin and bovine serum albumin have an isoelectric point of 5.2 and 5,
	respectively. They present positive charges at lower pH where alginate exhibit negative
	charges at low pH. Therefore, complex coacervation occurred mainly at pH range between
	~1.6 and 4.2 where due to opposite charges of alginate and proteins (Gorji et al., 2018).
	Another complex coacervation using gelatin and alginate has also been largely studied for
	encapsulation. Alginate, negatively charged at low pH, can form a complex coacervate with

[START_REF] Gorji | Complex Coacervation of Milk Proteins with Sodium Alginate[END_REF]

worked on characterization of alginate, beta-lactoglobulin and bovine serum albumin interactions in solution. A protein-alginate ratio of 2:1 was used for this study. Being amphoteric proteins, gelatin for essential oil encapsulation. Heckert Bastos et al. (2020) reported an optimum ratio of 6:1 gelatin to sodium alginate and a pH of 4 for black pepper essential oil encapsulation with an efficiency up to 82%. Similar efficiency result (74%) has been reported for citronella essential oil

Table 4 557

 4 Advantages and limitations of principal encapsulation methods using alginate. 558

	Methods	Advantages

Acknowledgements

Funding : This work was supported by "Cookal Company" ; "Impact Biomolecules" project of the "Lorraine Université d'Excellence", funded by the ANR "Investissements d'avenir" [grant number 15-004].


PhD Christine Rotonda
email: christine.rotonda@univ-lorraine.fr

M J Brennstuhl

E Eby

C Tarquinio

Impacts of COVID-19 on population well-being: Results of a web survey conducted in France during the first quarantine in 2020

Keywords: well-being, COVID-19, quarantine, resilience, optimism, general population

This study aimed to evaluate the impact of COVID-19 pandemic on mental well-being observed during the first quarantine implemented in France in 2020. This online survey included 1,876 French volunteer participants who completed data on lifestyle habits during the period of confinement and self-reported levels of resilience, optimism and psychological well-being. We observed that the score of participants' psychological well-being was considerably lower among single participants, students, the unemployed and those facing risk of financial insecurity. The results of the multivariate analysis showed that low levels of psychological well-being were significantly associated with single status and male gender.

Higher scores for general peer support, optimism, resilience and confidence in information shared within immediate circles of friends of family were also significantly positively associated with better mental health. Taken together, the results from this study showed that quarantine measures impacted the psychological well-being of the participants in our sample, that the degree of impact was strongly linked with individual levels of resilience and optimism, and that general support together with confidence in one's immediate circle of friends or family also played an important role in overall mental well-being.

Introduction

The coronavirus disease 2019 was first identified in Wuhan, China in December 2019 and spread internationally to become a major public health concern within weeks [START_REF] Phelan | The Novel Coronavirus Originating in Wuhan, China: Challenges for Global Health Governance[END_REF][START_REF] Zhu | A Novel Coronavirus from Patients with Pneumonia in China, 2019[END_REF]. Although virus mortality rates outside of China were initially quite low, suggesting that the health impact of the disease in other countries could potentially be minor, coronavirus cases grew rapidly. On March 12, 2020, the World Health Organization (WHO) declared the emergence of COVID-19 a public health emergency of international concern, the highest level of alarm under international law, and announced a global pandemic. Beginning on March 15, 2020, unprecedented measures were adopted in France to control COVID-19 virus transmission by limiting contact between individuals. To this end, a national level stay-at-home order was implemented on March 17, 2020 that lasted for a total of 55 days and was lifted on May 11, 2020. The objectives were twofold: to isolate those who tested positive for the virus by providing a context for care in a protected environment, and to reduce transmission of the virus by isolating asymptomatic carriers during the incubation period when the virus is most contagious. The novelty of this approach in a historical context since the 19 th century, notably compared to periods when quarantines were implemented during massive population flows, is that it combined citywide quarantine orders aimed at curbing the spread of the virus by restricting movement with additional response strategies such as isolating contagious persons receiving care in public or private designated healthcare facilities and, importantly, enforcing strict stay-at-home orders. The combined intiative that would eventually include regional travel restrictions and international border closures has affected entire populations across numerous countries.

A literature review conducted this year showed that extended stay-at-home quarantine measures could potentially have long-term consequences on psychological health [START_REF] Brooks | The psychological impact of quarantine and how to reduce it: Rapid review of the evidence[END_REF]. This review involved 24 studies conducted in ten countries on the psychological effects of quarantine during previous epidemics, and showed that extended periods of quarantine can trigger multiple psychological reactions including hyper-responsiveness to stress [START_REF] Yoon | System effectiveness of detection, brief intervention and refer to treatment for the people with post-traumatic emotional distress by MERS: A case report of community-based proactive intervention in South Korea[END_REF], irritability and insomnia [START_REF] Reynolds | Understanding, compliance and psychological impact of the SARS quarantine experience[END_REF]. Three of the included studies showed a correlation between longer duration of quarantine and poorer mental health, specifically an increase of negative emotions (such as fear and anger), misuse of psychoactive substances and post-traumatic stress symptoms [START_REF] Brooks | The psychological impact of quarantine and how to reduce it: Rapid review of the evidence[END_REF][START_REF] Hawryluck | SARS control and psychological effects of quarantine, Toronto, Canada[END_REF].

The primay objective of this article is to study the effect of variables most significantly associated with psychological well-being as measured during the first quarantine in France implemented in the spring and early summer of 2020. Indeed, the range of psychological repercussions of home confinement appear to be conditioned by the multiple ways home confinement is experienced: voluntary or imposed, brief or prolonged duration, confinement to the home environment or elsewhere, experienced alone or in a small group or family setting, and the presence of associated stress and/or traumatic factors, etc. [START_REF] Auxéméry | Le confinement généralisé pendant l'épidémie de Coronavirus: Conséquences médico-psychologiques en populations générales, soignantes, et de sujets souffrant antérieurement de troubles psychiques[END_REF]. Our intial hypothesis was that home confinement, its impact on financial security and inciting feelings of insecurity, would increase levels of stress experienced by the French population, perhaps more so than fear of the virus itself. We also theorized that individuals' optimism and resilience would correlate with levels of psychological well-being.

The Great East (Grand Est) region in France was one of the first in France to be strongly impacted by the COVID-19 pandemic. Several indicators included in the National Public Health Agency's report showed that the epidemic reached a peak in this region during the week of March 23 to 29, 2020, and had passed by April 10, 2020 (GrandEst_PER_COVID19_20200409.Pdf, n.d.). We therefore decided it was worth investigating possible differences in subjective measurements as compared to other regions of France during the same period.

Method

Study design and population

This cross-sectional observational study relied principally on an online questionnaire made available from April 3, 2020 to the evening of May 10, 2020 by the project leader, the Pierre Janet Center of the University of Lorraine, who posted it on their website and relayed it to social networks. All participants received detailed information describing the purpose of the study prior to completing the questionnaire. The survey was purposefully designed to be anonymous to ensure confidentiality and reliability of the data. All procedures were carried out in accordance with the French General Data Protection Regulation (GDPR).

Data collection

The questionnaire consisted of five parts:

-Part 1 : Questions about socio-demographics to collect data on respondent age, gender, marital status, with or without children, number of dependents per household, type of occupation, and occupational status during quarantine.

-Part 2: Questions concerning activities and immediate surroundings related to the quarantine environment, region of residence, potential number of occupants during quarantine, means of communication used during quarantine (telephone, social networks, phone messaging, video conferencing, etc.).

-Part 3: Questions about specific areas of concern such as virus contamination and lack of protection (masks, hydro-alcoholic gels, social distancing) for both oneself and one's immediate circle of family and/or peers (relatives, spouse, parents, children, friends, neighbors, colleagues, caregivers or support staff), occurrence of COVID-19 contamination and/or death related to COVID-19 within the immediate family or peer environment, risk of insecurity induced by the quarantine situation, perceived levels of support measured on a 10-point visual analog scale (VAS), trust in information provided by the government related to advances in research against spread of the COVID-19 virus, trust in information transmitted by social networks, by the press or circulated within the immediate circle of family and peers. A final set of questions included in this part pertain to general feelings of stress levels before and during quarantine.

Several variables in this section were aggregated to obtain the following scores:

-An insecurity score: this score is composed of 8 items. It was obtained by summing up the scores for general stress respondents felt before the quarantine was declared (a 10-point scale ranging from 0none to 10-extreme), the 4 scores pertaining to levels of concern about lacking adequate protection, and the 10 scores related to general levels of concern (based on the general population). This provided us with a score ranging from 0 (not at all concerned) to 150 (extremely concerned).

-A press confidence score which is the average of the 3 confidence scores (a 10-point scale ranging from 0-none to 10-extreme) related to information made available by printed press, television and radio.

-A confidence score related to information from family and friends which is the average of the 2 confidence scores (a 10-point scale ranging from 0-none to 10-extreme) in information from family and friends.

-Part 4: Three self-reported questionnaires validated in French measuring respondents' levels of optimism, well-being and resilience were administered:

1) The Warwick-Edinburgh Mental Well-Being Scale (WEMWBS), developed by [START_REF] Tennant | The Warwick-Edinburgh Mental Well-being Scale (WEMWBS): Development and UK validation[END_REF] and translated by Trousselart et al. (2016). This self-administered questionnaire, first developed in the United Kingdom for assessing a population's mental well-being, is comprised of 14 positively worded items covering hedonic and eudaimonic aspects of mental health. It uses 5 response categories for measuring the frequency of different states (0=never, 5=all the time) that are summed to provide a single score. The theoretical range of scores for WEMWBS is 14-70, with higher scores indicating a higher level of mental well-being.

2) The Life Orientation Test-Revised (LOT-R), validated in French, was used to measure optimism [START_REF] Trottier | Validation de la version canadienne-française du Life Orientation Test-Revised[END_REF]. Optimists approach life differently than pessimists in that they have positive expectations regarding the future [START_REF] Scheier | Distinguishing optimism from neuroticism (and trait anxiety, self-mastery, and self-esteem): A reevaluation of the Life Orientation Test[END_REF][START_REF] Scheier | Optimism, coping, and health: Assessment and implications of generalized outcome expectancies[END_REF]. Pessimists, on the other hand, will expect negative experiences in the future. The LOT-R was developed to assess individual differences in generalized optimism versus pessimism. It consists of 6 items, each scored on a 4-point Likert scale from "strongly disagree" to "strongly agree" and four filler items. Half of the coded items are phrased in an optimistic way to focus on an individual's expectations of postive outcomes, while the other half are phrased in a pessimistic way and focus on expectations of negative outcomes. Total scores can range from 0 to 24 indicating a maximum level of optimism.

3) Finally, the Brief Resilience Scale (BRS), developed by [START_REF] Smith | The brief resilience scale: Assessingthe ability to bounce back[END_REF] is a 6-item questionnaire designed to assess resilience as the self-perceived ability to bounce back or recover quickly from stress. It was translated and validated in French by [START_REF] Jacobs | Psychometric properties of the French Brief Resilience Scale[END_REF]. Each item is measured on a 5-point Likert scale (1=strongly disagree, 5=strongly agree). The three negatively worded items 2, 4 and 6 were recoded such that a high score indicates a high degree of resilience.

Calculating the BRS score is therefore carried out by assigning the value (from 1 to 5) to the responses for the 6 items. This sum ranging from 6 to 30 and then divided by the total number of completed items provides the final score. This score ranges from 1 to 5 and is interpreted as follows: a score of 1.00 to 2.99 indicates resilience; a score of 3.00 to 4.30 indicates normal resilience and a score of 4.31 to 5.00 indicates high resilience.

Analyses

As a first step, we conducted a descriptive analysis of the collected data using means and standard deviation for numeric variables and percentages for categorical variables. The internal consistency of the insecurity score composed of 8 aggregated items was then verified by calculating Cronbach's Alpha coefficient.

A variable corresponding to the regional deparment of residence (regions outside of the Great East Region versus those within the Great East Region) and a comparison of the collected variables was made according to this new variable.

Our main objective was to give specific focus to studying psychological well-being of our respondents during this first quarantine period in France. To do this, we compared each mental well-being score against each variable and a bivariate, and perfomed multivariate regression to explain this score.

Variables with p ≤ at 0.3 in bivariate analysis were integrated in to the multivariate model following a stepwise procedure. The scores were nevertheless presented in their raw format to interpret the strength of association of scores corresponding to optimism, resilience and insecurty, confidence and overall levels of mental well-being, for which the latter we linearized from 0 to 100 exclusively for results of the bivariate and multivariate linear regressions. Variables with p<0.05 were considered significantly associated with mental well-being.

Statistical analyses were perfomed using R Statistical Software.

Results

Sample Characteristics (Table 1)

A total of 1,876 individuals responded to our online questionnaire. These respondents were mainly women (82.3%), the average age among participants was 40 years, 64.9% of the participants were in a relationship, and 54% were parents. Concerning professional occupation, 75% reported being actively employed, 41.7% worked remotely during the quarantine period and 63% lived in the Great East region. Also included in our sample were medical and paramedical professionals (14%). Concerning the conditions of quarantine measures, 18% of the participants experienced stay-at-home orders alone in their homes and 15% had to no access to an outside environment. While participants in our sample reported few COVID-19 related deaths among immediate friends and family (4.3%), cases of the COVID-19 virus among the immediate circle was relatively high (58.5%), of which 5% of participants described impacted them personally. Finally, 16% of the participants indicated that they felt the quarantine would very likely threaten their financial security. A comparison of these characteristics related to place of residence (Non-Great East region vs. Great East region) showed several significant differences such as higher number of COVID-19 related deaths in the the Great East region (2.7% vs. 5.2%, p=0.021) and a higher proportion of participants indicating a risk of financial insecurity related to the quarantine in the Non-Great East region (p<0.001), but we also noted that a higher percentage of participants in the Non-Great East region were indepentently active professionals (p<0.001).

Remote means of communication during the quarantine period were also used with greater frequency (see Table 2). We noted that face-to-face contact with others was completely suspended for 61% of the participants. Use of remote means of communication (telephone, sms messaging, communication via social networks and video conferencing) increased significantly during the quarantine for all types of communication with the most significant evolution observed for video conferencing, for which use went from 18% daily or weekly before the quarantine to 64% during the quarantine. People with no social contact or less than one contact per week, regardless of the means of communication, represented less than 2% of our sample (n=23), 50 participants (2.7%) had only 1 contact per week and 78% of participants reported several contacts per week or even every day.

Description of scores (Table 3)

The raw scores for psychological well-being, optimism, resilience, insecurity, and confidence are described in Table 3, and our comparison of the raw psychological well-being scores for categorical variables is presented in Table 4.

We observed that the mean psychological well-being score across the sample was 49.6 (SD=7.8). As shown in Table 4, this score was lower among single participants (48.2; p<1.001), those confined to their homes alone (47.6; p<1.001), without access to the outdoors (47.7; p<0.0001), and among students and those with no professional activity (46.3 and 46.4 respectively; p<0.001) and people who indicated a risk of financial insecurity (47.9; p<0.001).

The level of psychological well-being was also significantly different (p<0.0001) according to the frequency of social contact, for all means of communication combined (telephone, sms messaging, video conferencing, social media, etc.): the mean score for those with no social contact or less than one time per week was 35.9 (SD=12), the mean score for those with one social contact per week was 47.0 (SD=7) and the average score for psychological well-being for those with several social contacts per week or every day was 49.9 (SD=7.5).

For the other scores, the average level for optimism across our sample was 12.8 (SD=4.5), the average score for resilience was 3.1 (SD=0.8), indicating normal resilience, and the average VAS score for overall perceived support was 5.4 (SD=2.4). The score for insecurity showed good internal consistency (Cronbach's alpha=.077). The mean insecurity score across our sample was 89.7 (SD=29.2). Finally, regarding confidence levels and corresponding scores, 54.5% of the participants expressed confidence in information provided by the governement and 68.3% expressed confidence in scientific research and researchers working towards understanding the COVID-19 virus and finding a vaccine. With regard to confidence levels measured on a scale of 0 to 10, the average confidence score for among participants was 2.5 (SD=2.2) for information found on social networks, the average score for confidence in information provided by the immediate circle of friends/relatives was 4.5 (SD=2.2), and the average score for confidence in the press was 5.0 (SD=2.2). A comparison of all scores by region of residence showed that only the security score was significantly different. Indeed, this score was significantly higher among people living in the Great East region (8.4 vs. 92.7; p<1.001).

Variables associated with levels of pscyhological well-being during quarantine (Table 4)

The results of the multivariate analysis showed that low psychological well-being during the quarantine period was significantly associated with single marital status (OR=-1.6; p=0.04) and male gender (OR=-2.2; p<0.02). High scores for perceived general support (=0.6; p<0.001), optimism (=0.27; p<0.001), resilience (=0.21; p<0.001), and confidence in information from the immediate circle (=0.04; p=0.004) were also positively associated with better mental well-being. Other variables for which we observed strong bivariate correlation with improved mental well-being (see Table 3) such as older age (=0.14; p<0.001), having children (OR=3.9; p<0.001) and trust in government and research (OR=2.7; p<0.001 and OR=3.9; p<0.001 respectively) no longer appeared as multivariate model. Similarly, several variables that were strongly associated with lower levels of psychological well-being during bivariate quarantine also stopped being multivariate model, such as student status (OR=-6.9; p<.001) and retired status (OR=-6.8; p<.001) compared to actively employed status, experiencing quarantine alone at home (OR=-3.9; p<0.001) or expressing an expected risk of financial insecurity due to the quarantine measures (OR=-3.0; p<0.001).

Discussion

This cross-sectional study, launched 15 days after the first nationwide quarantine measures were implemented in France and completed at the end of the quarantine period, is based on data collected from 1,148 individuals to ascertain how the experience of this unprecented quarantine situation impacted psychological well-being. Concerning specific conditions of this initial phase of home confinement, 42% of participants in our study remained gainfully employed and worked remotely, 18% experienced home confinement alone at home, 15% had no outdoor access during the quarantine, and 63% lived in the Great East region, which compared to other regions in France showed the highest scores for insecurity. At the time of this study, the Great East region was one of the most severely impacted by the epidemic in terms of active COVID-19 cases, which strained public health services. Protection of health care workers, paramedics and first responders, both in circulation and in hospitals, was rapidly recognized as the higest priority. Hospital teams and practitioners working in more isolated clinical settings played an integral part in the response effort, often working beyond normal working conditions to uphold values of protecting human health and dignity; they exhibited unfailing courage and dedication to the community as a whole, oftentimes to the detriment of their own well-being. An added dynamic for many of these workers was the necessity to stay employed during this period, despite the risks involved with continuing to work in potentially unsafe environments. The stress factors for many health workers were significant and centered essentially on three major dynamics: societal pressure surrounding the increasing anxiety-provoking nature of events unfolding worldwide coupled with an intense focus on recovery from the crisis through a commitment to providing critical patients with quality care, professional pressure marked by unprecedented workload and other predictable coping mechanisms, and pressure associated with personal life through either fear of contaminating one's family or returning to solitary conditions during confinement after a full day or night of work. Available data on the pscyhological consequences on health workers during this global health situation show, for example, a prevalence of 14.5% anxiety, 8.9% depression and 7.7% posttraumatic stress disorder [START_REF] Tan | Psychological Impact of the COVID-19 Pandemic on Health Care Workers in Singapore[END_REF]. Similar data were observed in the population of nearly 1,000 frontline health professionals in Wuhan, China (initial focus of the pandemic).

In France, on average and excluding specific events, the score for psychological well-being of individuals measured by the same questionnaire we used in this study has previously been reported as 53 [START_REF] Franck | Covid-19 et détresse psychologique[END_REF], whereas we observed in our own study a lower average score of 3.4 points. This score was found to be even lower for single individuals, those who experienced quarantine home alone, those without access to the outdoors during quarantine, students, the unemployed and participants who indicated potential risk of financial insecurity. A large survey conducted in France in the spring of 2020 that involved 20,000 people reported similar results using the same questionnaire on psychological well-being [START_REF] Franck | Covid-19 et détresse psychologique[END_REF]. Social support was perceived to be strongly associated with mental well-being, where our own sample showed that 78% of the participants had some form of daily social contact, mainly by telephone, social networks and sms messaging.

Similarly, our results show lower optimism levels than other studies conducted on the general population [START_REF] Schou-Bredal | Population-based norms of the Life Orientation Test-Revised (LOT-R)[END_REF]. Finally, the resilience score considered as the ability to "cope" with a difficult or stressful situation, often traumatic, indicated that participants in our study exhibited adequate resilience capacity. These two scores, optimism and resilience, were found to be strongly associated with levels of psychological well-being across our sample. Individuals with higher levels of optimism are more inclined to believe that the goals they set are achievable and, as a result, they invest more effort in achieving their goals than pessimists, and most of them succeed [START_REF] Chang | Optimism and pessimism as partially independent constructs: Relationship to positive and negative affectivity and psychological well-being[END_REF][START_REF] Grover | Optimism and attribution about uncertain events: What happened to missing flight MH370?[END_REF]. Individuals with higher levels of optimism appear more inclinded to use effective coping mechanisms when faced with difficulties. They seek operational solutions and engage themselves both cognitively and emotionally in finding solutions, whereas pessimists are more inclined to use coping strategies that involve withdrawl, either mentally or behaviorally, from the given situation. It is therefore not surprising that in the context of the present global health situation, individuals with higher levels of optimism are better able to take care of themselves and implement more appropriate preservation and prevention strategies. This would also suggest that they are less inclined let themselves become overwhelmed by the situation for the fact that they take a proactive approach and responsbility for their own self care without waiting for outside help. Conversely, people with higher levels of pessimism can quickly become overwhelmed with feelings of powerlessness faced with persistent and omnipresnet bad news in the media as the pandemic unfolds. Resilience can be defined as the ability to adapt in the face of adversity and trauma or other significant stressors [START_REF] Norris | Looking for resilience: Understanding the longitudinal trajectories of responses to stress[END_REF]. It is the ability to succeed, to live and develop positively and in a socially acceptable way despite stress or adversity normally associated with risk of negative outcome.

Numerous studies have shown that psychological resilience can be seen as an intermediary between environmental stress and an individual's psychological state [START_REF] Hao | Relationship between resilience, stress and burnout among civil servants in Beijing, China: Mediating and moderating effect analysis[END_REF][START_REF] Howell | Relational resilience as a potential mediator between adverse childhood experiences and prenatal depression[END_REF] that can mitigate the negative effects of the former [START_REF] Poole | Childhood adversity and adult depression: The protective role of psychological resilience[END_REF][START_REF] Sheerin | The impact of resilience and subsequent stressful life events on MDD and GAD[END_REF]. Thus, resilience acts as an interface that allows people to protect themselves, including against psychological distress. In the limited number of studies that have examined the effect of resilience on mental health in the context of a global health crisis, resilience was found to be negatively correlated with both depression and anxiety in exposed individuals [START_REF] Osofsky | Deepwater horizon oil spill: Mental health effects on residents in heavily affected areas[END_REF]. Based on our own results, it appears that the combination of optimism and good resilience facilitates maintaining a certain level of well-being among the general population, which to our knowledge is rarely pointed out in studies on the effects of health crises on psychological health at the population level.

Although 80% of our sample was female, our multivariate analysis showed that the men's psychological well-being was significantly impacted. This result may be related to two contingent phenomena. The first being that it has been suggested that men are more at risk for contracting the virus than women, which may have contributed to levels of reported concern [START_REF] Rozenberg | Are we equal in adversity? Does Covid-19 affect women and men differently?[END_REF].

Secondly, an important aspect is that the quarantine measures imposed a situation men may, to some degree, experience differently than women; it consisted of staying at home and no longer working in a professional setting that is often linked to identity. It is worth noting here that our study was conducted during the first quarantine period in France when working remotely was a relatively new concept.

Many found themselves in situations of temporary unemployment due to a total loss of professional activity. Interestingly, we also noted a protective effect in the context of couples and, in a bivariate way, age. Indeed, young people and particularly students, often living away from their families and with limited financial means, experienced the most pronounced impacts in terms of psychological well-being, as shown in both our study and the literature [START_REF] Bourion-Bédès | Psychological impact of the COVID-19 outbreak on students in a French region severely affected by the disease: Results of the PIMS-CoV 19 study[END_REF]. This study showed that the global health situation during the first quarantine in France led 15.2% to perceive themselves as moderatly anxious and 9.8% as strongly anxious. Another study carried out among Chinese students revealed that 24.9% experienced anxiety as a results of the COVID-19 pandemic [START_REF] Cao | The psychological impact of the COVID-19 epidemic on college students in China[END_REF]. Similarly, a study involving Greek students during periods of quarantine reported an increase in anxiety scores, depression and suicidal thoughts, a general deterioration in quality of life and poorer sleep quality [START_REF] Kaparounaki | University students' mental health amidst the COVID-19 quarantine in Greece[END_REF].

It was generally noted that the elderly felt well protected by the health measures put in place and experienced the first quarantine in relative comfort. Age did not emerge as signifiantly associated in multivariate analyses: the average age of our sample was 40 years. In addition, retirees, who represented only 6% of our sample, reported psychological well-being scores similar to younger, working people.

During the first quarantine, 68% of participants reported feeling reassured by scientific knowledge and expressed that they felt confident that research would lead to solutions for overcoming the pandemic. This confidence was significantly associated with improved mental well-being but only in bivariate analyses. We noted that trust in information from immediate family and friends played a greater protective role in terms of psychological well-being, while this score remained average.

The main strength of this study is the sample size. Additionally, tools that were validated in French with good psychometric properties were used for describing individuals' mental health status.

Clearly, it would have been informative to recontact the participants during the second quarantine which was experienced differently by the population, and indeed, a useful line of questioning would have been what these differences were exactly. Were the individuals able to benefit from their first quarantine experience, or conversely, did the negative phenomena observed in terms of malaise, depressive or anxiety disorders increase? The question is equally pertient for those subjects with higher scores related to pscyhological well-being. There is nothing to suggest that such parameters would persist over the long term. Importantly, it was widely understood by everyone that the first quarantine period would eventually end and that the total duration would be a matter or weeks, possibly months. Virtually no one expected the possibility of being faced with a second or third quarantine or prolonged enforced stay-at-home orders. The consequences in terms of psychological health, family, social and professional life combined with economic impacts associated with such an abrupt shut-down of society for the greater good of public health come with a host of secondary effects, the dimension and scope of which no one could have anticipated; the challenge is that we cannot fully understand how the different parameters evalutated in this study will or could evolve in the medium, long or even very long term and what the impacts will be on the general public. The fact that our study was transversal but also completely anonymous prevents us from being able to contact participants for follow up evaluations; indeed, we felt anonymity of participants would serve the overall integrity of the data but we also did not anticipate a second quarantine. However, a cluster analysis would make it possible to to identify risk profiles and rather protective profiles. This analysis will be carried out soon by our team. A second limitation is the mode in which the questionnaire in our study was diffused to potential respondents, which was primarily through social networks and direct online responses. Despite advantages that come with using these methods (lower cost, speed of data collection, reduced data entry errors, greater freedom for the resondent) [START_REF] Ganassali | The Influence of the Design of Web Survey Questionnaires on the Quality of Responses[END_REF][START_REF] Stephenson | Studying Political Behavior: A Comparison of Internet and Telephone Surveys[END_REF], certain drawbacks nonetheless emerged from this approach. First and foremost, this type of data collection requires a certain familiarity with computers and the Internet, as well as access to social networks from which we disseminated the survey. Several authors mention that respondents to online surveys are generally regular, even expert, Internet users, a factor that can potentially impact the type of sample (generally more affluent, more educated and younger, for example) [START_REF] Bosnjak | Sample composition discrepancies in different stages of a probability-based online panel[END_REF]. Importantly, this aspect of our study design could call into question the representativeness of our sample.

Conclusion

This study allowed us to observe that the psychological well-being of participants was impacted during this quarantine period, that it was strongly associated with levels of both optimism and resilience, and that support in general together with trust in the environment also played an important role in levels of mental well-being. Our results are farily consistent with what has been reported in the literature in recent months, notably that quarantine measures have long-term consequences on the population's psychological well-being [START_REF] Brooks | The psychological impact of quarantine and how to reduce it: Rapid review of the evidence[END_REF]. This presents a constraint that governments and policy-makers have not fully acknowledged, despite the fact that a deeper understanding of this key dimension being the mental health of the general population could potentially act as a lever to helping or hindering society in efforts to forge paths to recovery and envision new ways of functioning to overcome this current global health crisis. If the emotional state of the population remains negatively affected and the health situation persists over longer periods, potentially several months as we are currently experiencing, it will be imperative to question the longterm effects of psychological health on physical health. It is indeed essential that we build on what has thus far been revealed by the current pandemic situation and its emotional impacts to establish a link between mental and physical states, particularly given that we now know that the negative emotional state is associated with chronic stress and chronic anxiety, both known to weaken the immune system, the body's primary defense against infection and disease [START_REF] Powell | Social stress up-regulates inflammatory gene in the leukocyte transcriptome via β-adrenergic induction of myelopoiesis[END_REF].

Declarations of Competing Interest

None

Table 1

 1 Sociodemographic description of the sample (n=1876) and comparison of these data by residential region relative to the Great East RegionStatistical tests performed: Wilcoxon rank-sum test; chi-square test of independence

	Total	Great East Region
	Respondents	No	Yes
	(N=1876)	(N=694 ; 37%)	(N=1182 ; 63% )	p-value*
	N (%)	N (%)	N (%)

Table 3

 3 Description of Scores for Mental Health, Insecurity and Confidence and Comparison by Region

	Total Respondents		Great East Region
	(N=1876)		No	Yes		p-
			(N=694 ; 37%)	(N=1182 ; 63% )	value*
	n (%)	mean (SD)	n (%)	mean (SD)	n (%)	mean (SD)

* Statistical tests performed: Wilcoxon rank-sum test; chi-square test of independence SD: Standard Deviation

This research did not receive any specific grant from funding agencies in the public, commercial, ornot-for-profit sectors