Marjorie Yon

Claire Billotey

Jean-Daniel Marty
email: marty@chimie.ups-tlse.fr

Gadolinium-based contrast agents: from gadolinium complexes to colloidal systems

Keywords: Magnetic resonance imaging, MRI, gadolinium, relaxivity, hybrid polyion complexes, gadolinium nanoparticles

Nowadays, most of the clinically used contrast agents are based on the use of gadolinium molecular complexes like Dotarem®, Magnevist®…. Nevertheless, developing new gadolinium based contrast agents with enhanced relaxivity properties while avoiding gadolinium release in human tissue is still of paramount importance. We describe here two promising families of contrast agents which meets these criteria and compared their properties to the ones of clinically used Gd complexes. The first one is based on contrast agents obtained by covalent or non-covalent interactions of gadolinium molecular complexes with polymers, nanoparticles or liposomes. The second family is based on the formation of gadolinium particles (Gd2O3, GdPO4, NaGdF4, GdF3...) of controlled morphology and composition. The performance of these two families is described as well as their development perspective.

Introduction

Magnetic resonance imaging (MRI) is a powerful medical diagnostic tool [START_REF] Mansfield | Line scan proton spin imaging in biological structures by NMR[END_REF] whose efficiency is greatly improved by the use of exogeneous contrast agents (CAs) [START_REF] Patten | Positive bowel contrast agent for MR imaging of the abdomen: phase II and III clinical trials[END_REF][START_REF] Caravan | Strategies for increasing the sensitivity of gadolinium based MRI contrast agents[END_REF]. The two most common classes of contrast agents are: (a) T1 or positive contrast agents that shorten proton longitudinal or spin lattice relaxation time and (b) T2 or negative contrast agents that shorten proton transversal or spin-spin relaxation time [START_REF] Young | Enhancement of relaxation rate with paramagnetic contrast agents in NMR imaging[END_REF]. Whereas superparamagnetic iron oxide nanoparticles are most widely studied as T2 agents, CAs based on the lanthanide ions Gd 3+ are most often employed as T1 agents. Indeed, gadolinium, a lanthanide metal ion with seven unpaired electrons, has demonstrated its efficiency in enhancing proton relaxation because of its high magnetic moment and paramagnetic properties and its ability to coordinate water efficiently. However, the toxicity of Gd ions related to their substitution or antagonist activity of calcium in a variety of cellular reactions [START_REF] Das | Effects of lanthanum in cellular systems[END_REF] or in the occurrence of nephrogenic systemic fibrosis [START_REF] Sieber | Gadolinium-based contrast agents and their potential role in the pathogenesis of nephrogenic systemic fibrosis: the role of excess ligand[END_REF] prevents its direct administration. Therefore to limit the cytotoxicity of Gd ions in commercially available CAs, Gd ions are chelated by specific interacting structures like linear multifunctional or macrocycles structure (Figure 1). The first families of Gd-based CAs were using linear complexing molecules. In 1987, Gddiethylenetriamine pentaacetic acid (Gd-DTPA, MAGNEVIST®) [START_REF] Harpur | Preclinical Safety Assessment and Pharmacokinetics of Gadodiamide Injection, a New Magnetic Resonance Imaging Contrast Agent[END_REF][START_REF] Weinmann | Characteristics of gadolinium-DTPA complex: A Potential NMR contrast agent[END_REF] was proposed for clinical uses. Then three other linear Gd-based CAs were further approved until 2004: Gd-DTPA, gadodiamide -OMNISCAN®) (Radbruch, A. et al., 1987[START_REF] Schmiedl | Comparison of initial biodistribution patterns of Gd-DTPA and albumin-(Gd-DTPA) using rapid spin echo MR imaging[END_REF], gadopentetate dimeglumine (OPTIMARK®) and gadobenate dimeglumine (MultiHance®), [START_REF] Johansson | Gadobenate Dimeglumine (Multihance) in Mr Angiography: An In-Vitro Phantom Comparison with Gadopentetate Dimeglumine (Magnevist) at Different Concentrations[END_REF]. In addition, macrocyclic Gd-based CAs (gadoteridol, Gd-tetraazacyclododecanetetraacetic acid and gadobutrol) were also developed to limit the release of free Gd ions and were authorized by several regulatory health agencies since 1994 as commercial products (PROHANCE ® , DOTAREM ® , GADAVIST ® ). Interestingly, these macrocyclic Gd-based CA enables to observe a lower level of remaining Gd in vivo in comparison to their linear counterpart (Gibby W.A. et al., 2004). Nevertheless, molecular complexes possess limitations, such as residual toxicity [START_REF] Kanda | High signal intensity in the dentate nucleus and globus pallidus on unenhanced T1-weighted MR images: relationship with increasing cumulative dose of a gadolinium-based contrast material[END_REF], Kanda et al., 2015, Radbruch et al., 2015) and reduced efficiency at the higher magnetic fields of modern MRI instruments. This compels the main regulatory health agency in 2017 to control more carefully the use of this kind of contrast agents and European medicine agency suspended the authorization of the intravenous linear Gd-based CAs.

Current challenge is to develop new Gd-based CAs that avoid the release of the free Gd ions to prevent further complications. At the same time there is still a need of Gd-based CAs with enhanced relaxation properties to decrease the amount of injected compound. To reach such a goal colloidal CAs are of special interest. Indeed the use of Gd-based colloidal structures in the 10 to 100 nm size range enable first to increase the relaxivity of obtained CAs. This effect is related to a much slower tumbling effect compared to molecular gadolinium complexes induced by a huge increase in the molecular weight of the gadolinium species (and of hydrodynamic radius). Slowing tumbling induces higher relaxivity. Gd ions located within the colloidal species will rotate at the same (and very low) rate than the whole NPs, increasing the observed relaxivity. Therefore lower injected levels are permitted. Moreover, colloidal structures present some additional features concerning their stability that make them candidate of choice for new families of CAS. In this review, we will present two different families of enhanced colloidal Gd-based CAs (Figure 2): 1) gadolinium complexes grafted or in interaction onto colloidal structures, 2) inorganic nanoparticles of controlled shape and properly functionalized. As can be seen Gd ions are interacting specifically with chelating agents in the form of macrocycles or linear ligands. Whereas those chelating agents enable to minimize ion exchange with competing ions, they also tend to decrease the amount of water molecules in direct interaction with Gd. Hence, at a same magnetic field value of 0.5T, relaxivity of free gadolinium is about 7 mM -1 .s -1 against 3.8 mM - 1 .s -1 when the ion is chelated by DTPA through specific interaction, at 37°C [START_REF] Villaraza | Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function and Pharmacokinetics[END_REF]. This results in a relative poor efficiency of this CAs. Moreover, 90% of the product is out of the body in less than an hour through renal filtration and vessel leakage, which let short time to these species to act as CAs and necessitate the injection of high amount of CAs. The aim thus is to optimize the pharmacokinetic of Gd-based CA and to increase the relaxivity value of these CAs. To this purpose, chelating agents are grafted to polymer chains, colloids or lipids. Immobilization of Gd-complexes enable to reduce the tumbling rate by slowing down rotational motions and thus provides higher relaxation times. [START_REF] Du | Facile marriage of Gd3+ to polymercoated carbon nanodots with enganced biocompatibility for targeted MR/fluorescence imaging of glioma[END_REF]. The attachment of several complexes on macromolecules, colloids or lipids thereby increases relaxivity value and thus permits to decrease the administration dose. [START_REF] Ding | Functional Surface Engineering of C-Dots for fluorescent Biosensing and in vivo Bioimaging[END_REF][START_REF] Du | Facile marriage of Gd3+ to polymercoated carbon nanodots with enganced biocompatibility for targeted MR/fluorescence imaging of glioma[END_REF]. For this, after the protection of 4 of its 5 carboxylic acid functions, protected DTPA was reacted with the amine functions located on the surface of carbon nanodots (Figure 3). After a deprotection step, the modified particles complexed gadolinium ions to form a colloidal contrast agent. These supported CAs had several advantages compared to Gd-DTPA complexes: increased relaxivities (r1 value of 8 mM -1 •s -1 compared to 4 mM -1 •s -1 for the Gd-DTPA at a 3T magnetic field) and an ability to interact more easily with cancer cells thanks to enhanced permeability and retention effect. In addition the fluorescence of the carbon nanodots provided another way to image the glioma, making the CAs dual-imaging species.

Figure 3. Preparation of modified carbon nanodots with clinical Gd-DTPA in a two-step process: 1) coupling of amino group on the surface of carbon nanodots with protected DTPA (cDTPAa) thanks to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling agent and deprotection 2) complexation with gadolinium ions. Adapted from references [START_REF] Du | Facile marriage of Gd3+ to polymercoated carbon nanodots with enganced biocompatibility for targeted MR/fluorescence imaging of glioma[END_REF].

Another strategy to obtain Gd complexes grafted onto colloidal structures was described by [START_REF] Tran | Functionalization of Gadolinium Chelates Silica Nanoparticle through Silane Chemistry for Simultaneous MRI/ 64 Cu PET Imaging[END_REF] by using a DOTA properly modified with a terminal triethoxy group as illustrated in Table 1. Siloxane nanoparticles covered by DOTA were then obtained by using sol gel chemistry. At a 1.5 T magnetic field, these particles had higher longitudinal relaxivity value than for free Gd-DOTA complexes, i.e r1=18 mM -1 .s -1 and 4.2 mM -1 .s -1 respectively. Further association with 64 Cu enabled to use these particles as multimodal agents for MRI and PET imaging techniques. The biodistribution revealed that they were rapidly eliminated in the kidney.

Relaxation properties of gadolinium complexes can also be improved by grafting them onto macromolecules. Hence, [START_REF] Younis | MRI-visible polymer based on poly(methyl methacrylate) for imaging applications[END_REF] grafted Gd-DTPA complex on poly(methyl)methacrylate-co-propargylmethacrylate to obtain Gd-DTPA-PMMA nanoparticles (see structure in Table 1). Different amounts of Gd-DTPA were incorporated with the PMMA polymer chains. This enable to test the influence of the Gd concentration on the contrast obtained at a 7T magnetic field. Interestingly, magnetic resonance images showed that the contrast was not significantly improved by increasing the amount of gadolinium and that low amount of gadolinium ions were sufficient to obtain good contrast properties. In addition these modified polymers showed a good stability through low gadolinium leakage in PBS buffer at 37°C during 90 days. [START_REF] Li | Macromolecular Ligands for Gadolinium MRI Contrast Agents[END_REF] studied the influence of macromolecular architecture on relaxivity with linear, hyperbranched and star copolymer grafted with DO3A contrast agent (See Figure 4A). As shown in Figure 4B, a significant increase of relaxivity was measured for grafted Gd-DO3A complex compared to the relaxivity of free complex whatever the architecture of polymer is. Moreover the authors evidenced the role of each architecture on molecular tumbling and water exchange rate. They highlighted that, in the case of the star shape copolymer, the polymer shell reduced the diffusion of water molecules near the core-located Gd complexes which induced a decrease of relaxation rate compared to linear or hyperbranched architectures. Hence, despite the globular aspect of the hyperbranched structure, the mobility of gadolinium ion inside the macromolecular architecture was the same than for the linear one, proving the very low influence of the hydrodynamic radii of the molecule. In addition to the architecture, average molecular weight of the chosen polymer can also play a role on relaxation properties. To study this, [START_REF] Margeruma | Gadolinium(III) DO3A macrocycles and polyethylene glycol coupled to dendrimers -Effect of molecular weight on physical and biological properties of macromolecular magnetic resonance imaging contrast agents[END_REF] studied polyamido(amine) (PAMAM) dendrimers of different generation of a structure comprising Gd-DO3A CAs. An increase of relaxivity was observed when the molecular weight of the polymer increased due to slower molecular tumbling: from 14.8, to 16.9 and 18.8 mM -1 ⋅s -1 for PAMAM polymer of 3 rd ; 4 th and 5 th generation respectively. However [START_REF] Bryant | Syntehsis and relaxometry of high-generation (G=5, 7, 9, and 10) PAMAM dendrimer-DOTA-gadolinium chelates[END_REF] evidenced a saturation limit for PAMAM of 7 th generation and explained this phenomenon with the stagnation of the water exchange rate.

In order to enhance relaxivity properties, apart from nanoparticles and polymer structures, Gd-complexes can also be covalently linked to lipid. These modified lipids can be inserted in liposome structures made of phospholipids that presents low toxicity. [START_REF] Kabalka | Gadoliniumlabeled liposomes: targeted MR contrast agents for the liver and spleen[END_REF] have introduced modified Gd-DTPA (see structure in Table 1) in liposome structures with an overall size of 440 nm. As expected, a higher relaxivity is observed for liposomic structures compared to GD-DTPA. Interestingly, the ability to insert increasing amount of Gd complex in the liposome structure enabled to measure an increase of relaxivity of the vesicular specie up to a content of 33,3%. The biodistribution of these contrast agents indicated that the liposomes are rapidly removed from blood and degraded in the liver by Kupffer cells. Another interesting application of those structures is to combine different functionalities like luminescence, magnetic or targeting in one particle. Skupin-Mrugalska et al. ( 2018) combined a model photosensitizer for photodynamic therapy, zinc phtalocyanine, and a MRI Gd-based contrast agent based on Gd-DTPA modified with a lipophilic chain. Studies proved that Gd did not hamper the photosensitizing ability of the zinc specie. Moreover, comparisons between liposomes comprising or not zinc phtalocyanine showed that the presence of the photosensitizer enhance relaxivity properties of these Gd-based CAs. At low magnetic field (16,5 MHz) high r1 values up to 50 mM -1 ⋅s -1 were found, whereas at high magnetic field (400 MHz), r2 values up to 90 mM -1 ⋅s -1 were measured. Lastly, liposomes can be considered as nanocarriers, which can participate to active targeting strategies by a lipophilic chain bearing a specific probe. Furthermore, the following of this targeting with MRI device would inform on the pathway of the agent towards the target. To this end, [START_REF] Yoo | Gadolinium-Functionalized Peptide Amphiphile Micelles for Multimodal Imaging of Atherosclerotic Lesions[END_REF] designed fibrin-binding, peptide amphiphile micelles by incorporating at different concentration a targeting peptide cysteine-arginine-glutamic acid-lysine-alanine (CREKA) with amphiphilic molecules containing Gd-DTPA complex. Besides, the targeting particles were compared to non targeting ones. Whereas in vitro studies confirmed that a uniform amphiphile backbone and micelle shape homogeneity had a large contribution to the clot-targeting capability, in vivo studies showed fibrin specificity was conferred by the peptide ligand without much difference between the nanoparticle formulations.

From gadolinium specific complexes to hybrid polyion complexes

Hence, immobilization of Gd-chelates onto macromolecules or nano-objects is one efficient way to provide more efficient relaxation by slowing down rotational motion, although the synthetic pathways to such modified polymers can be complicated. An alternative to these strategies is to induce the formation of aggregates (also called coacervates or hybrid polyion complexes, HPICs) between metal ions and block copolymers bearing more simple but nonspecific functional groups (Figure 5). This was done for example by [START_REF] Wang | Controlled mixing of lanthanide(III) ions in coacrevate core micelles[END_REF] where a negatively charged Gd complex interacted with a diblock cationic copolymer. Another alternative is the use of uncomplexed gadolinium ions which will interact with polymers functionalized by multidentate groups (Figure 5). [START_REF] Wang | Stable polymer micelles formed by metal coordination[END_REF][START_REF] Wang | Lanthanide-Dipicolinic Acid Coordination Driven Micelles with Enhanced Stability and Tunable Function[END_REF][START_REF] Bryant | Syntehsis and relaxometry of high-generation (G=5, 7, 9, and 10) PAMAM dendrimer-DOTA-gadolinium chelates[END_REF] Figure 5. Related strategies leading to the formation of CAs from the interaction of A) charged block copolymers with lanthanides complexes [START_REF] Wang | Controlled mixing of lanthanide(III) ions in coacrevate core micelles[END_REF] or B) block copolymers comprising multidentate complexing groups with lanthanide ions. [START_REF] Wang | Stable polymer micelles formed by metal coordination[END_REF]. Adapted from references [START_REF] Wang | Controlled mixing of lanthanide(III) ions in coacrevate core micelles[END_REF] and [START_REF] Wang | Stable polymer micelles formed by metal coordination[END_REF].

Nevertheless, both examples involve gadolinium-chelating groups with complex structures. New, less complex strategies could allow the easy development of a new generation of CAs. Thus, HPICs obtained from the addition of simple charged polyvalent metal ions (e.g. Cu 2+ , Zn 2+ ) to a solution of double hydrophilic block copolymers with generic functions that are not specifically designed for metal ions could be of great interest. Recently it was demonstrated that gadolinium-containing HPICs prepared (see Figure 6) from a commercially available diblock copolymer made of poly(acrylic acid), PAA, and poly(ethylene oxide), PEO, blocks exhibited exceptionally high stability upon dilution. Adapted from reference (Frangville et al., 2016). Indeed, at a magnetic field of 7T, the hybrid polyion complex, with a mean diameter ca 20 nm, exhibited a longitudinal relaxivity of 15.4 mM -1 ⋅s -1 , against 4.1 mM -1 ⋅s -1 in the same conditions for Gd-DOTA and 8.8 mM -1 ⋅s -1 for hybrid gadolinium oxide nanoparticles. Moreover, the in vivo study showed a pretty long blood remanence, followed by a fast urinary elimination. These systems were combining a low gadolinium quantity with a high relaxation signal, meeting with the initial requirements based on a limited release of free gadolinium within the tissues and enhanced relaxation properties (Frangville et al., 2016;[START_REF] Mingotaud | Agents de contraste pour imagerie medicale[END_REF].

Inorganic Nanoparticles.

In order to minimize the in vivo release of gadolinium ion, many studies have proposed the use of gadolinium-based nanoparticles. In addition there are numerous advantages to nanoparticle-based agents including easy surface modification for incorporating specific vectors and the opportunity to combine properties to give multimodal agents. (Cheng Y. et al., 2014;[START_REF] Cheng | Gelatin-encapsulated iron oxide nanoparticles for platinum (IV) prodrug delivery, enzymestimulated release and MRI[END_REF]. Hence, recent reports have described NPs of gadolinium oxides [START_REF] Bridot | Hybrid gadolinium oxide nanoparticles: multimodal contrast agents for in vivo imaging[END_REF][START_REF] Park | Paramagnetic ultrasmall gadolinium oxide nanoparticles as advanced T1 MRI contrast agent: account for large longitudinal relaxivity, optimal particle diameter, and in vivo T1 MR images[END_REF][START_REF] Das | Gadolinium Oxide Ultranarrow Nanorods as Multimodal Contrast Agents for Optical and Magnetic Resonance Imaging[END_REF][START_REF] Faucher | Rapid synthesis of PEGylated ultrasmall gadolinium oxide nanoparticles for cell labeling and tracking with MRI[END_REF], fluorides [START_REF] Evanics | Water-Soluble GdF3 and GdF3/LaF3 Nanoparticles -Physical Characterization and NMR Relaxation Properties[END_REF][START_REF] Xu | Gadolinium fluoride mesoporous microspheres: Controllable synthesis, materials and biological properties[END_REF] or phosphates [START_REF] Lessing | Synthesis and characterization of gadolinium phosphate neutron absorber[END_REF][START_REF] Fang | Systematic synthesis and characterization of single-crystal lanthanide orthophosphate nanowires[END_REF][START_REF] Hifumi | Gadolinium-based Hybrid Nanoparticles as a Positive MR Contrast Agent[END_REF][START_REF] Hifumi | Dextran Coated Gadolinium Phosphate Nanoparticles for Magnetic Resonance Tumor Imaging[END_REF][START_REF] Ren | Mn2+ dopant-controlled synthesis of NaYF4:Yb/Er upconversion nanoparticles for in vivo imaging and drug delivery[END_REF][START_REF] Dumont | DNA surface modified gadolinium phosphate nanoparticles as MRI contrast agents[END_REF][START_REF] Rodriguez-Liviano | Synthesis and properties of multifunctional tetragonal Eu :GdPO4 nanocubes for optical and magnetic resonance imaging applications[END_REF]. However, the use of these nanoparticles as contrast agents is still dependent on the production of nanoparticles of well controlled size and morphology, the relaxation properties being highly dependent on these parameters as has been suggested for Gd2O3 NPs although with different surface coatings [START_REF] Park | Paramagnetic ultrasmall gadolinium oxide nanoparticles as advanced T1 MRI contrast agent: account for large longitudinal relaxivity, optimal particle diameter, and in vivo T1 MR images[END_REF]. Moreover, the particles require suitable coatings, such as organic ligands or polymers to improve their biocompatibility, reduce their toxicity and raise the interaction with water molecules. .

Control of nanoparticle size and effect on relaxivity properties.

As just mentioned, the control of NPs size is required to have access to NPs with reproducible relaxivity property. Therefore a tremendous amount of work was dedicated to the development of synthetic method to get access to stable nanoparticle with controlled size and morphology. Gadolinium oxides (Gd2O3), fluorides (GdF3, NaGdF4) or phosphates (GdPO4) have been described in literature, are obtained by precipitation reaction in presence of well-chosen stabilizing/agent. These hybrid materials offer relaxivities per mole of Gd comparable or superior to the common molecular agents. Among them, gadolinium phosphate nanoparticles (GdPO4 NPs) are of special interest because of the combination of low solubility, ease of surface modification, and the variety of synthetic routes available to form particles. [START_REF] Liu | Rare earth and yttrium phosphate solubilities in aqueous solution[END_REF] Hydrothermal synthesis [START_REF] Hifumi | Gadolinium-based Hybrid Nanoparticles as a Positive MR Contrast Agent[END_REF][START_REF] Hifumi | Dextran Coated Gadolinium Phosphate Nanoparticles for Magnetic Resonance Tumor Imaging[END_REF][START_REF] Ren | Mn2+ dopant-controlled synthesis of NaYF4:Yb/Er upconversion nanoparticles for in vivo imaging and drug delivery[END_REF][START_REF] Laudise | Hydrothermal Synthesis of Sapphire[END_REF][START_REF] Hikichi | Syntheses of Rare Earth Orthophosphates[END_REF] and more recently microemulsion synthesis [START_REF] Dumont | DNA surface modified gadolinium phosphate nanoparticles as MRI contrast agents[END_REF] or a templated mineralization process [START_REF] Du | Facile preparation and bifunctional imaging of Eu-doped GdPO4 nanorods with MRI and cellular luminescence[END_REF] were used to form well-defined GdPO4 NPs as nanorods or nanowires of tens to hundreds nanometers length. Interestingly, nanoparticles of GdPO4, or even particles with GdPO4 modified-surfaces, have been shown to be compatible with biological media. For example, [START_REF] Li | Surface Interactions with Compartmentalized Cellular Phosphates Explain Rare Earth Oxide Nanoparticle Hazard and Provide Opportunities for Safer Design[END_REF] demonstrated that pretreatment of rare earth oxide nanoparticles with phosphate, protects against in vivo biological interference of gadolinium ions with Ca 2+ involved in cell metabolism as well as pro-fibrogenic effects favored by the presence of Gd 3+ ions. This result was further supported by cell toxicity and in vivo experimental assays performed with dextran-coated GdPO4NPs that showed no impact on cell viability, observed over long residence times in plasma. [START_REF] Hifumi | Gadolinium-based Hybrid Nanoparticles as a Positive MR Contrast Agent[END_REF][START_REF] Hifumi | Dextran Coated Gadolinium Phosphate Nanoparticles for Magnetic Resonance Tumor Imaging[END_REF]Synthesis pathways to obtain nanometrically controlled particle sizes have thus been developed through the use of different polymer families of different architectures: block polymers, branched polymers. These polymers act as both a growth limiter during the growth process of nanoparticle and a stabilizer to provide increased colloidal stability in biological environments: As a first step, Gd 3+ was sequestered within the polymer and then GdPO4 NPs were obtained after a precipitation step induced by the addition of phosphate ions. In these conditions final NPs structure will depend strongly on the polymer architecture and on the concentration of reactant in solution. Playing with the mixing speed also allows to obtain particles of a better controlled size. In that context ionic nanoprecipitation is of special interest (Figure 7): it involves the rapid mixing of a water stream containing Gd 3+ ions with one containing stabilizing agent and phosphonate ions in a confined impinging jet mixer or multi-inlet vortex mixer. Rapid mixing assures homogeneous supersaturation prior to nucleation, growth and aggregation events, and the diffusion-limited assembly driven by ionic interactions results in particles with narrow size distributions. The particle growth is arrested by the adsorption of the polymer onto the NP surface resulting in the formation of a dense, stabilizing corona. (Pinkerton et al., 2017) GdPO4 and GdxEu1-xPO4 NPs in the 20 to 100 nm range were thus obtained with relaxivity values r1 and r2 in the 15-40 mM -1 ⋅s -1 range at 1.4 T.

Figure 7. Left : Principle of the iFNP process, with the hydrophilic block copolymer in green and blue. Right : TEM images of GdPO4 NPs obtained through nanoprecipitation process. Adapted from reference (Pinkerton et al., 2017).

Moreover to determine the relationship between particle size and relaxivity values, it is also necessary to be able to obtain particles of variable size but functionalized in an equivalent way. [START_REF] Lu | Size-tunable NaGdF4 nanoparticles as T2 contrast agents for high-field magnetic resonance imaging[END_REF] reported a simple and feasible approach to regulate NaGdF4 NPs growth by changing the density of trisodium citrate chelator to obtain different sizes of NPs in the range of 100-220 nm. These nanoparticles of controlled size were then covered with a silica shell of similar thickness using a reverse microemulsion method. R1 and r2 relaxivity measurements of these NaGdF4@SiO2 NPs were investigated in water at 0.5 T and 7 T. At 0.5 or at 7 T, r1 relaxivities decreased with the increase of particle size whereas r2 values increased dramatically. Hence at 0.5 T particles with a diameter of 120 nm showed r1 = 1.4 mM -1 ⋅s -1 and r2 = 4.36 mM -1 ⋅s -1 whereas particles with a diameter of 250 nm showed r1 = 0.39 mM -1 ⋅s -1 and r2 = 8.55 mM -1 ⋅s -1 . Interestingly the observed effect on r2 was far more pronounced at higher field (7T). In addition, these NaGdF4@SiO2 NPs showed good compatibility and low cytotoxicity. This example demonstrated the primary interest in controlling particle size. Other examples proposed chemical recipes to obtain size controlled NPs with equivalent organic coating down to a few nm. Size-controlled GdPO4 nanowires in a range of 120±39 nm down to 6±2 nm were synthesized in presence of hyperbranched polymers based on the polyamidoamine HyPAM in aqueous solutions and at room temperature. [START_REF] Frangville | Hyperbranched polymer mediated size-controlled synthesis of gadolinium phosphate nanoparticles: colloidal properties and particle sizedependence on MRI relaxivity[END_REF] As shown on Figure 8, relaxivity measurements of these hybrid materials showed a maximum for the 23 nm particle sample and revealed an optimal length at ca. 23±11 nm for which promising r1 and r2 values were obtained (r1= 55±9 mM -1 ⋅s -1 ; r2= 67±11 mM -1 ⋅s -1 ). Hence a size dependence on measured r1 and r2 is observed with lower values of relaxivity for both larger and smaller particles. Similar observations of an optimal particle size were observed for other particle systems, (Park et al., 2013;[START_REF] Wang | Controlled mixing of lanthanide(III) ions in coacrevate core micelles[END_REF] including NaGdF4 and Gd2O3, and was attributed to contributing factors influencing relaxivity that have opposite particle size corelations. One influencing factor is the availability of Gd 3+ ions at the surface, as direct chemical exchange of water molecules is the largest contributor to proton relaxation. The surface to volume ratio (S/V) increases as particle size gets smaller, accounting for the most often observed increase in relaxivity as particles become smaller. On the other hand, correlation times and therefore tumbling times are another factor. [START_REF] Villaraza | Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function and Pharmacokinetics[END_REF][START_REF] Caravan | Strategies for increasing the sensitivity of gadolinium based MRI contrast agents[END_REF][START_REF] Johnson | Size-Tunable, Ultrasmall NaGdF4 Nanoparticles: Insights into Their T1 MRI Contrast Enhancement[END_REF][START_REF] Wiener | Dendrimer-based metal chelates: A new class of magnetic resonance imaging contrast agents[END_REF] Tumbling times depend on the medium viscosity and hydrodynamic volume of the particles. If we assume the viscosity does not change for the different particle suspensions, then for a reasonable range of particle sizes, relaxivity should increase with hydrodynamic volume. For this serie with the same chemical coating, the apparent hydrodynamic diameter follows the same linear trend as the increase of NP length from ~10 to ~100 nm, allowing us to consider the NP length as the key parameter. These opposing contributions of surface area and hydrodynamic volume are then expected to give rise to an optimum particle size for any set of conditions. It is important to realize that correlation times will be affected by other parameters, such as medium viscosity and applied magnetic field strength, so any 'optimum' particle size will depend on the application. Furthermore, since the tumbling time depends on the hydrodynamic volume of the composite polymer-particle object, the polymer mediated preparation affords the potential for tuning the response through synthetic variation of both the inorganic particle size and the thickness of the polymer coating.

Critical role of coating on relaxivity properties.

As we have just seen, the control of the nature and size of the inorganic part is of crucial importance to have access to nanoparticles with optimal relaxation properties for a given application. A second element playing on relaxivity values is the nature of the organic part covering the particles. The organic part surrounding the inorganic core performs several functions above its ability to control NPs growth during NPs synthesis.

Its primal role is to ensure that the particles have good colloidal stability in order to avoid any aggregation or precipitation phenomenon in vivo. For this purpose the use of organic shell comprising poly(ethylene glycol) (PEG) is of special interest as it minimizes adsorption of biomolecules onto the NPs and avoids fast clearance from blood system. Hence many works described the use of this polymer to cover inorganic NPs. For instance Lakew Mekuira et al. ( 2017) synthesized gadolinium oxide NPs covered with a dendrimer whose external functions were modified by PEG. The presence of PEG chains enhanced the biocompatibility, the circulation time in blood and the colloidal stability of the contrast agent. The leakage of Gadolinium ion was checked through absorbance measurements and also by titrating in PBS buffer for 120 hours and only a non-significant amount of Gd ions was released from this hybrid material. In addition, relaxivity values measured at a 7T magnetic field were very high for both r1 and r2 (r1=53.9 mM -1 ⋅s -1 and r2=182.8 mM -1 ⋅s -1 ) and compared very favorably to the one measured for Gd2O3 nanoparticles covered by different functionnalized agent like PVP or single PEG chain (for these two systems relaxivities values equal to r1=12.1 mM -1 ⋅s -1 and r2=33.2 mM -1 ⋅s -1 and r1=4.4 and r2=28.9 mM -1 ⋅s -1 were found respectively).

In addition to stability property, this organic part enables to control the accessibility of water molecules in the vicinity of gadolinium and to confer colloidal stability to the hybrid material systems. Zheng et al. described the synthesis by a coprecipitation method of GdF4 NPs with uniform morphology and narrow size distribution (8.6±1.1 nm). Then by using a two-step ligand exchange strategy, these nanoparticles were covered by different polymeric ligands: poly(ethylene glycol (PEG), polyéthyleeimine (PEI) and polyacrylic acid (PAA) [START_REF] Zheng | Gd-Dots with Strong Ligand-Water Interaction for Ultrasensitive Magnetic Resonance Renography[END_REF]. As illustrated in Figure 9, longitudinal (r1) and transverse (r2) relaxivities were found higher when NPs were covered by PAA group. Moreover when the average molecular weight of PAA used is changed (1200, 2000, 5000 or 8000 g⋅mol -1 ) optimal relaxivity values were found for PAA2000. This result evidenced that strong surface ligand-water interacWon can efficiently promote relaxivity. It is thus possible to develop ultrasmall Gd-dots with higher relaxation performance than CAs with bigger size. The called Gd-Dots were about 2.1 nm by size and then were more exposed to water, which explains the highest relaxivity value compared to other bigger nanoparticles. The promoting effect can be ascribed to the formation of second hydration shell, where the increased number of exchangeable water molecules and their prolonged residence time result in an enhanced second-sphere contribution.

Figure 9. A. Longitudinal (r1) and transverse (r2) relaxivitiesof the samples measured at 0.5 T. Relaxivities of different samples measured at 0.5 T, including Gd-DTPA, PAA2000-capped NaGdF4NPs (8.6 and 3.2 nm), and Gd-dots (2.1 nm). DLS analysis of the samples. Adapted from [START_REF] Zheng | Gd-Dots with Strong Ligand-Water Interaction for Ultrasensitive Magnetic Resonance Renography[END_REF].

Consequently, the choice of the structure of the organic part covering inorganic particles is of prior importance to obtain stable CAs with long residence time in vivo and high relaxivity values.

Toward multimodal agents.

By playing on both the nature of the inorganic and organic parts it is thus possible to obtain agent NPs with multi-modal imaging or targeting properties. Nanoclusters containing both gold NPs and gadolinium ions were thus used as contrast agent for different imaging techniques Computed Tomography (CT), Near-Infrared Fluorescence (NIRF) and Magnetic Resonance (MR) imaging [START_REF] Hou | Nanoparticles for multi-modality cancer diagnosis : simple protocol for self-assembly of gold nanoclusters mediated by gadolinium ions[END_REF]. These nanoclusters were stable through time with a hydrodynamic diameter of about 200 nm and the measured longitudinal relaxivity value r1 was about 25 mM -1 ⋅s -1 , which is about 5 times the one of the Gd-DTPA. Other examples described the introduction of luminescence properties on GdPO4 NPs either by inserting Europium ions during the precipitation of gadolinium in presence of phosphate (Figure 10A) or by covering GdP04 NPs with luminescent molecules like indocyanine green or polymers bearing a fluorescent moiety (Figure 10B). [START_REF] Pinkerton | Quench ionic flash nano precipitation as a simple and tunable approach to decouple growth and functionalization for the one-step synthesis of functional LnPO4-based nanoparticles in water[END_REF] Interestingly in the former case changing Gd/Eu ratio enabled also to enhance relaxivity properties of obtained NPs: higher relaxivity values were obtained with lower gadolinium content (Figure 10A). Another combination of magnetic sensitivity and luminescence property was described by [START_REF] Maldiney | Gadolinium-Doped Persistent Nanophosphors as Versatile Tool for Multimodal In Vivo Imaging[END_REF] who worked on nanocrystals based on trivalent chromium-doped zinc gallate. Indeed, in the aim to get multimodal agents, Gd was added to these nanomaterials to both follow the luminescence of the agent and evaluate its relaxation properties. The nanoparticles were doped with Gd and showed higher relaxivity values with increasing Gd content (up to 4 mol%). With the idea to increase the residence time of the nanoparticles in the body, these ones were functionalized with poly(ethylene glycol) (PEG) chains through hydroxylation, condensation with amino-silanes and grafting of PEG5kDa-NHS. This functionalization resulted in a higher contrast compared to a reference mouse. Additionally, it was evidenced that the uptake from the Kupffer cells in the liver was delayed, according to MR images, confirming the longer residence time. Last but not least, active targeting remains one of the main applications of the synthesis of Gd-based nanoparticles. The contrast agents become then theranostic agents, used to give information about a precise area in the body, which is targeted. [START_REF] Ahmad | Cyclic RGD-Coated Ultrasmall Gd2O3 Nanoparticles as Tumor-Targeting Positive Magnetic Resonance Imaging Contrast Agents[END_REF] grafted on Gd2O3 nanoparticles with an average diameter about 2 nm, 5 commercial targeting probes composed of Arg-Gly-Asp peptides varying by the kind of amino acids present on the peptide chain, further named cyclic-RGDs. Grafting yield was between 40 and 50% and hydrodynamic diameter of the obtained nanohybrids was about 6 nm. Measured longitudinal relaxivities at 0.47 T and 25°C varied between 10 and 18 mM -1 ⋅s -1 which compared favorably to the ones of Gd-DTPA (r1 = 3.8 mM -1 ⋅s -1 ) and Gd-DOTA (r1 = 4.2 mM -1 ⋅s - 1 ). This is explained with a stronger hydrophily of the cyclic-RGDs, raising the interaction with water molecules, and from a high quantity of Gd ions the surface of nanoparticle. In vitro internalization of one of the cyclic-RGD was made in U87MG tumor cells, showing the aggregation of nanoparticles on the cell surface after 24h of incubation. Finally in vivo experiments highlighted a higher contrast in tumoral areas compared to normal and necrosis parts, proving the efficient targeting of these nanoparticles towards tumor cells.

Conclusion.

Developing new gadolinium based contrast agents is of tremendous importance with the aim to drastically limit the quantity of Gd ions remaining within the tissues and two challenges have to be tackled: decreasing the amount of Gd ions released and/or increasing the relaxivity properties of the Gd based CAs to limit the amount of injected compoundIn this context, colloidal CAs are of special interest. Indeed the use of Gd-based colloidal structures in the 10 to 100 nm size range enables first to increase the relaxivity values of obtained CAs thanks to slower tumbling effect. The grafting or interaction of gadolinium complexes with other chemical structures such as nanodots, polymer with different architectures, liposomes or hybrid polyion complexes, helps to the formation of colloidal structures with improved relaxation properties. Furthermore, using inorganic nanoparticles (Gd2O3, GdPO4, NaGdF4, GdF3) instead of Gd complexes strengthens the stability of Gd within formed nanoparticle and thus limit the release of Gd ions in vivo and thus empowers the performance of Gd-based CAs at high magnetic fields. In addition, it is noteworthy to keep in mind that the properties of the inorganic particles are still dependent on the production of nanoparticles with well controlled size, morphology and appropriate coating. Lastly, playing with the composition of colloidal structures enables the insertion of additional features (targeting, fluorescence,…) that made them candidate of choice for new families of CAS with multimodal properties.

Figure 1 .

 1 Figure 1. Gd-DOTA, Gd-DTPA, Gd-HP-DO3A and Gd-DTPA-BMA chemical structures. Adapted from (Hermann et al., 2008).

Figure 2 .

 2 Figure 2. Main families of Gd-based contrast agent.

Figure 4 .

 4 Figure 4. A. Chemical structure of linear, hyperbranched and star polymers P(OEGA-co-PFPA) with grafted DO3A contrast agents. B. Longitudinal relaxivity value for the single chelating agent and for the three architectures of the dendrimer. Adapted from reference[START_REF] Li | Macromolecular Ligands for Gadolinium MRI Contrast Agents[END_REF].

Figure 6 .

 6 Figure 6. A. The addition of gadolinium ions to a double hydrophilic block copolymer forms polymeric nanoparticles called hybrid polyion complexes HPICs(in yellow-orange: ionizable block; in dark blue: neutral block). B. Brain angiography images versus time obtained with contrast agent based on HPICs compared to Gd-DOTA clinically available contrast agent.Adapted from reference(Frangville et al., 2016).

Figure 8 .

 8 Figure 8. GdPO4/HyPAM size dependence on r1 and r2 relaxivities (1.4 T). Particle lengths were determined before dialysis. Lines are guides to the eye. Adapted from (Frangville, Gallois et al, 2016).

Figure 10 .

 10 Figure 10. A. Relaxitiy values r1 and r2 of GdEuPO4 NPs as a function of the Gd/Eu ratio. B. Structure of fluorescent polymer used as a functionalizing agent and picture of the resulting excited solution. Adapted from (Pinkerton et al., 2018).

Table 1 .

 1 Table 1 presents some typical examples of CAs developed by using such a strategy. Typical examples of gadolinium complexes grafted on colloidal strutures. We will describe here some typical examples of the main families of colloidal systems. For a more complete description, Villaraza et al. (2010) reviewed the manifold types of colloidal systems (e.g. dendrimers, micelles, proteins, liposomes) tailored with Gd complexes. Du et al. described the grafting of DTPA complexes onto carbon nanodots

	Structure	r1 relaxivity in	Size	Refer
		mM -1 .s -1	(nm)	ence
		(magnetic field)
		8.05 (3T)	100	Ding et al., 2014
		17.97 (1. 5T)	4,5	Tran et al., 2018
				Younis et
		NG	NG	al., 2016
		Linear
		15.6	NG
		(0,5T)
		Hyperbranched
				Li et al.,
		15,4	8-9	2012
		(0,5T)
		Star
		13,5
		(0,5T)

Acknowledgements. The authors gratefully acknowledge the CNRS, University Paul Sabatier and region Occitanie for financial support.


Sophie Yvon

Lauriane Schwebel

Loubnah Belahcen

Helene Tormo

Magali Peter

Djamila Ali Haimoud-Lekhal

Hélène Eutamene
email: helene.eutamene@inra.fr

Gwenaelle Jard

Effects of thermized donkey milk with lysozyme activity on altered gut barrier in mice exposed to water-avoidance stress

Keywords: donkey milk, lysozyme activity, gut, pasteurization

Nutrition plays a crucial role in human gut health through the improvement of gut barrier functionality. Donkey milk represents an interesting source of natural antimicrobial factors such as lysozyme. Recently, anti-inflammatory properties of donkey milk lysozyme activity were described in a mouse model of ileitis. The current increase of donkey milk consumption highlights the necessity to propose a healthy milk compliant with microbiological standards. This study aims to define a heat treatment of donkey milk, retaining its high lysozyme activity, and to evaluate its beneficial effects on a gut barrier impairment model due to chronic stress in mice. To perform this experiment, samples of raw donkey milk were collected in 15 distinct French farms. Microbiological analysis and lysozyme content and activity were evaluated for each sample. Then, several heat treatments were carried out to define a time and temperature combination that allowed for both a reduction in the number of total micro-organisms, increasing the shelf-life of the product, and preservation of lysozyme activity. The beneficial effect of heated donkey milk on the gut barrier of mice was evaluated and compared with raw donkey milk. We found that samples of raw donkey milk showed low total mesophilic microbial counts, and no pathogens were detected. Among the different heat-treatment procedures tested, a 2-min, 72°C combination was determined to be the most optimal time and temperature combination to preserve lysozyme activity and increase the shelf-life of donkey milk. Oral administration of this heat-treated donkey milk in mice counteracted chronic stress-induced intestinal damage, illustrated by gut hyper-permeability and low-grade inflammation, similar to raw donkey milk. We have demonstrated for the first time that oral intervention with donkey milk, optimally heat-treated to retain enzymatic lysozyme activity, improves intestinal barrier damage linked to psychological stress in mice.

INTRODUCTION

Donkey milk (DKM) represents a significant source of natural protective antimicrobial factors [START_REF] Šarić | Antibacterial properties of domestic Balkan donkeys' milk[END_REF]Cosentino et al., 2015a[START_REF] Cosentino | Effects of different heat treatments on lysozyme quantity and antimicrobial activity of jenny milk[END_REF][START_REF] Labella | Optimization and validation of a chromatographic method for quantification of the lysozyme in jenny milk[END_REF]. It has been shown that DKM has ability to improve human health [START_REF] Brumini | Whey proteins and their antimicrobial properties in donkey milk: A brief review[END_REF][START_REF] Aspri | Donkey milk: An overview on functionality, technology, and future prospects[END_REF]. For example, DKM can be useful in cases of cow milk allergy [START_REF] Carroccio | Intolerance to hydrolysed cow's milk proteins in infants: Clinical characteristics and dietary treatment[END_REF], in the prevention of atherosclerosis [START_REF] Tafaro | Immunological properties of donkey's milk: its potential use in the prevention of atherosclerosis[END_REF], and in the positive stimulation of the immune system [START_REF] Jirillo | Donkey's and goat's milk consumption and benefits to human health with special reference to the inflammatory status[END_REF]. All these relevant healthy effects seem to be related to the biochemical composition of DKM. Whey proteins represent 35 to 50% of its nitrogen fraction, with 2 g/L of lactoferrin and 1 to 4 g/L of lysozyme [START_REF] Guo | Composition, physiochemical properties, nitrogen fraction distribution, and amino acid profile of donkey milk[END_REF]. The fraction of DKM containing lysozyme exerts anti-tumoral activities in vitro [START_REF] Mao | Anti-proliferative and anti-tumour effect of active components in donkey milk on A549 human lung cancer cells[END_REF]. Recently, we showed that daily DKM consumption with constant lysozyme activity (LZA) has anti-inflammatory effects in a mouse model of ileitis. This effect is highly dependent on LZA and is associated with normalization of antimicrobial peptide levels in Paneth cells. These data suggest that DKM must contain high LZA in order to obtain beneficial effects on gut mucosa (Yvon et al., 2016).

Nutrition plays a crucial role in human gut health through its effects on gut microbiota and barrier func-tionality [START_REF] Gottlieb | Impact of human aging and modern lifestyle on microbiota[END_REF][START_REF] Marco | Health benefits of fermented foods: Microbiota and beyond[END_REF]. Homeostasis is bound to the interactions of commensal bacteria and host immunity. A disruption of this homeostasis is found in several pathologies, such as inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS; [START_REF] Ma | Nutrients mediate intestinal bacteria-mucosal immune crosstalk[END_REF]. Low-grade intestinal mucosal inflammation is described in IBS patients and associated with increase in gut permeability and visceral pain [START_REF] Zhou | Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome[END_REF][START_REF] Vivinus-Nébot | Functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation[END_REF]. In humans, stressful life events exacerbate these gut disorders [START_REF] Lerebours | Stressful life events as a risk factor for inflammatory bowel disease onset: A population-based case-control study[END_REF][START_REF] Moloney | Stress and the microbiota-gut-brain axis in visceral pain: Relevance to irritable bowel syndrome[END_REF]. In mice, chronic stress, such as water-avoidance stress (WAS), induces disruption of the colonic epithelial barrier, promotes gut micro-inflammation, induces hypersensitivity to colorectal distension, and creates dysbiosis [START_REF] Bradesi | Repeated exposure to water avoidance stress in rats: A new model for sustained visceral hyperalgesia[END_REF][START_REF] Ferrier | Impairment of the intestinal barrier by ethanol involves enteric microflora and mast cell activation in rodents[END_REF][START_REF] Larauche | Stress-related alterations of visceral sensation: Animal models for irritable bowel syndrome YVON ET AL[END_REF][START_REF] Watanabe | Chronic psychological stress disrupted the composition of the murine colonic microbiota and accelerated a murine model of inflammatory bowel disease[END_REF].

The presence of antimicrobial components such as lysozyme, immunoglobulins, and lactoferrin in raw DKM keeps total levels of bacteria low [START_REF] Pilla | Hygienic and health characteristics of donkey milk during a followup study[END_REF][START_REF] Cavallarin | A survey on the milk chemical and microbiological quality in dairy donkey farms located in NorthWestern Italy[END_REF][START_REF] Aspri | Donkey milk: An overview on functionality, technology, and future prospects[END_REF], and therefore confers a long natural shelf life [START_REF] Zhang | The antimicrobial activity of donkey milk and its microflora changes during storage[END_REF]. Processed in other foodstuffs, DKM acts to prevent bacterial growth and effectively extends their shelf life (Cosentino et al., 2015b). However, contamination with Enterococcus faecium and Enterococcus faecalis, strains that are pathogenic in humans, has been detected in raw DKM [START_REF] Verraes | A review of the microbiological hazards of raw milk from animal species other than cows[END_REF][START_REF] Aspri | Raw donkey milk as a source of Enterococcus diversity: Assessment of their technological properties and safety characteristics[END_REF]. Responsible for an estimated 80% of Enterococcus infections [START_REF] Kayser | Safety aspects of enterococci from the medical point of view[END_REF], E. faecalis is a strain adaptable to different substrates and resistant to both pasteurization (30 min at 62°C; [START_REF] Kearns | Nosocomial enterococci: Resistance to heat and sodium hypochlorite[END_REF] and antimicrobial LZA [START_REF] Hébert | Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance[END_REF]. The recent expansion of donkey breeding and the increased consumption of DKM sold on farms for direct consumption highlight the necessity for farmers to offer a safe and biologically effective DKM to consumers. Heat treatments with appropriate time and temperature combinations are used in the production of milk to obtain a safe and suitable end product. A heat treatment of 30 min at 62.5°C is currently applied to human breast milk [START_REF] Sousa | Effect of thermal pasteurisation and high-pressure processing on immunoglobulin content and lysozyme and lactoperoxidase activity in human colostrum[END_REF][START_REF] Van Der Voorn | Stability of Cortisol and cortisone in human breast milk during Holder pasteurization[END_REF]. However, this time and temperature combination causes a drastic loss of up to 44% of LZA [START_REF] Sousa | Effect of thermal pasteurisation and high-pressure processing on immunoglobulin content and lysozyme and lactoperoxidase activity in human colostrum[END_REF].

This study aimed (1) to establish an overview of lysozyme levels and activity in microbiologically characterized French DKM; (2) to develop a low-cost heattreatment process to render DKM compliant with microbiological standards and preserve its LZA; and (3) to test the ability of this heated milk, compared with raw DKM, to counteract the gut hyper-permeability and micro-inflammation induced by chronic stress in mice.

MATERIALS AND METHODS

Experimental Design

This study comprised 3 consecutive phases, diagrammed in Figure 1. The first consisted of a diagnosis phase to describe the bacterial load and the lysozyme levels and activity in 15 raw DKM samples collected from 15 different French farms. The next phase consisted of optimizing the thermization process on 1 selected milk, obtained from La Ferme du Hitton (Biran, France), chosen for the farm's high quality of rearing and animal well-being. First, several heat treatments were carried out to define a time and temperature combination that would both reduce the number of total microorganisms and preserve LZA in DKM (step 1). Second, linked to the low level of microorganisms contained in the selected DKM, an enrichment with endogenous bacteria and an artificial contamination with the thermotolerant strain E. faecalis were performed (step 2). Then, the best thermization treatment process for generating safe milk, compliant with microbiological standards, was selected from the suitable heat treatments previously tested (step 3). On this basis, an aging test was performed to characterize the shelf life of the thermized DKM (step 4). In the last phase of the study, the beneficial effects of pasteurized DKM were evaluated in an in vivo model of chronic stress in mice, which promotes gut hyper-permeability and a microinflammation response.

Donkey Milk Sample Collection

Donkey milk samples were provided from 15 different farms across France. After milking, the samples were collected in sterile flasks, immediately frozen, transported to the laboratory under frozen conditions, and stored at -18°C until analysis.

Microbiological Analysis

Microbiological Load. Total mesophilic bacteria, lactic acid bacteria, and thermotolerant coliforms were counted to evaluate the health quality of the different raw DKM coming from the 15 French farms. One milliliter each of undiluted samples and dilutions of 10 -1 , 10 -2 , and 10 -3 of each sample were used for counting. All these analyses were performed in triplicate. The media and the incubation conditions are described in Table 1. Tests for pathogens (Listeria monocytogenes, Salmonella spp., Escherichia coli, and coagulase-positive Staphylococcus) were conducted in all the samples by Socsa Analyse (L'Union, France). The results were expressed in log 10 cfu/mL.

Microbial Enrichment and Artificial Contamination of Selected DKM.

Linked to the low level of microorganisms counted in the selected DKM from La Ferme du Hitton, an enrichment of endogenous bacteria and an artificial contamination with E. faecalis were performed. Briefly, a bacterial smear from donkey milk growth on PCA medium was enriched in a brainheart medium incubated overnight at 37°C, and several aliquots were stored at -20°C with cryoprotectant. Then, DKM was enriched to reach 6.3 log 10 cfu/mL of total bacteria in milk. Artificial contamination was performed by adding 7.3 log 10 cfu/mL E. faecalis strain (EI-Purpan collection, Toulouse, France) to DKM. Using the different media described in Table 1, total bacteria and E. faecalis bacteria were counted before and after the heat treatments tested that did not alter the enzymatic LZA.

Aging Tests of

Lysozyme Levels and Activity

Concentration of Lysozyme. Levels of lysozyme were determined using the reversed-phase (RP)-HPLC approach adapted from [START_REF] Herrouin | New genetic variants identified in donkey's milk whey proteins[END_REF] and [START_REF] Vincenzetti | Donkey's milk protein fractions characterization[END_REF]. From each milk sample, 20 mL was taken and skimmed by centrifugation at 3,000 × g for 30 min at 32°C. Whole caseins were precipitated from the skim milk by adjusting the pH to 4.25 with HCl. After centrifugation, the supernatant samples corresponding to the whey proteins were dialyzed against distilled water at 4°C overnight using an Interchim membrane (Montluçon, France) and then freeze-dried and stored at -18°C until analysis The flow rate was 1 mL/min. The proteins eluted from the RP-HPLC column were monitored at 280 nm by a UV detector (Ultimate 3000 RS diode array detector) included in the HPLC system (Thermo Fisher Scientific). The amount of lysozyme was determined using a calibration curve obtained from a standard chicken egg-white lysozyme prepared in Buffer A and expressed in milligrams per milliliter. Activity of Lysozyme. Activity of lysozyme was assessed using an EnzCheck Lysozyme Assay Kit (Molecular Probes E-22013; Thermo Fisher Scientific). The LZA levels reflected by the fluorescence intensity were measured by a spectrofluorometer (Tecan SAS, Lyon, France) and quantified via a calibration curve obtained with a standard chicken egg-white lysozyme. The LZA was expressed in units per milliliter.

Thermal Treatments

To optimize the heat treatment, 10 distinct fresh raw milk samples from La Ferme du Hitton were used. After milking, samples were collected in sterile flasks, immediately transported to the laboratory at 4°C, and heated in a water bath at several combinations of time and temperature (Table 2). Then, samples were rapidly cooled by immersion into cold water. Before and after each heat treatment, LZA was measured. The total bacteria and thermotolerant bacteria E. faecalis were counted after each heat treatment.

In Vivo Experiments

Animals. Eight-week-old male C57BL/6 mice (weight 23 ± 0.5 g; Janvier Labs, Le Genest-Saint-Isle, France) were used. Mice were maintained in pathogenfree animal facilities at a constant temperature (21 ± 2°C) and a 12-h light/dark cycle. Mice had free access to water and standard rodent pellets (containing 18.6% CP, 6.2% crude fat, 5.3% crude ash, and 3.5% crude fiber; Harlan Sprague Dawley Inc., Indianapolis, IN). To minimize environmental stress, animals were handled for 1 week before the experiments. Animal care and work protocols, as well as sacrifice procedure, were approved by the local ethical committee, according to EU Directive 2010/63/EU (Agreements #TOXCOM_0143HE and APAFiS #4972-2016021814258245).

Water-Avoidance Stress. Briefly, mice were placed on a 3 × 3-cm platform fixed in the center of a 40 × 40-cm pool filled with cold water 1 h per day for 9 d [START_REF] Bradesi | Repeated exposure to water avoidance stress in rats: A new model for sustained visceral hyperalgesia[END_REF]. Each WAS sessions was performed between 0800 and 1000 h. Control mice were submitted to a similar procedure using an empty pool (sham stress).

Lipocalin-2 Levels. Lipocalin-2 (LCN-2) is a neutrophil gelatinase-associated lipocalin (NGAL) contained in neutrophil granules, used as a biomarker of intestinal inflammation. To quantify LCN-2, mouse feces were collected and subsequently mixed in PBS for 20 min to obtain a homogeneous fecal suspension [START_REF] Chassaing | Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation[END_REF]. Then the samples were ground using FastPrep (MP Biomedicals, Illkirch, France; 3 times for 15 s each at 6 m/s, with 1-min breaks on ice) and centrifuged for 10 min at 10,000 × g at 4°C. Clear supernatants were collected, and samples were diluted in the kit-recommended reagent diluent (1.0% BSA in PBS). We then measured LCN-2 levels using the Duoset murine LCN-2 ELISA kit (R&D Systems, Minneapolis, MN). The obtained values were normalized by the weight of fecal material.

Gut Permeability. Fluorescein isothiocyanate (FITC) is a small (389 Da) fluorescent molecule present in plasma after an oral administration of FITC. This plasma fluorescence reflects gut permeability status [START_REF] Inczefi | Congenitally elevated gut permeability is linked to basal visceral hyposensitivity but stress-induced visceral hypersensitivity in CA-MLCK mice[END_REF][START_REF] Edelblum | The microbiome activates CD4 T-cell-mediated immunity to compensate for increased intestinal permeability[END_REF]. Mice received 250 µL of free FITC (1 mg/mL, F7250; Sigma-Aldrich, Saint-Quentin-Fallavier, France) by oral route. Three hours after FITC administration, a blood sample was taken from the facial submandibular vein and centrifuged at 10,000 × g for 10 min. Clear supernatants were collected, and samples were diluted in sterile saline solution (NaCl 0.9%, Sigma-Aldrich).

The fluorescence levels present in the plasma were assessed using a TECAN fluorimeter (485 excitation/528 emissions), and the obtained values were normalized by the BW of each mouse. The values were expressed as percentage of difference from the control group. Experimental Protocol. Male C57BL/6 mice were divided into 6 distinct groups (n = 10 per group): 3 control groups and 3 WAS groups. Animals orally received a PBS solution (0.2 mL/d), raw DKM (0.2 mL/d), or pasteurized DKM (pasteurized for 2 min at 72°C; 0.2 mL/d, volume adjusted ±0.1 mL, so that each animal received 5,900 U of LZA, as in the raw DKM group), respectively, and the BW of each mouse was recorded daily. At the end of the WAS or the sham stress procedure, fecal materials were collected to evaluate the gut inflammatory response reflected by fecal LCN-2 levels, and the total gut permeability was assessed as previously described.

Statistical Analysis

Data were expressed as mean ± SEM. For statistical analysis, GraphPad Prism 4 (GraphPad, San Diego, CA) was used. For all experimental studies, multiple comparisons between treatments or animal groups were performed using 1-way ANOVA, followed by Bonferroni's multiple comparison test. Statistical significance was accepted at P < 0.05. To evaluate the correlation between LZA and lysozyme levels, a linear regression was performed to obtain the regression coefficient (R 2 ).

RESULTS AND DISCUSSION

Characterization of French Raw Donkey Milk: Microbiological Quality, Lysozyme Content, and Lysozyme Activity

Samples of French raw DKM showed low total mesophilic microbial counts (3.82 ± 1.56 log 10 cfu/mL). These values are close to the average range (2.40 ± 1.87 log 10 cfu/mL) reported in literature [START_REF] Herrouin | New genetic variants identified in donkey's milk whey proteins[END_REF][START_REF] Salimei | Composition and characteristics of ass's milk[END_REF][START_REF] Zhang | The antimicrobial activity of donkey milk and its microflora changes during storage[END_REF][START_REF] Pilla | Hygienic and health characteristics of donkey milk during a followup study[END_REF][START_REF] Sarno | Microbiological quality of raw donkey milk from Campania Region[END_REF][START_REF] Cavallarin | A survey on the milk chemical and microbiological quality in dairy donkey farms located in NorthWestern Italy[END_REF][START_REF] Malissiova | Assessment of donkey milk chemical, microbiological and sensory attributes in Greece and Cyprus[END_REF][START_REF] Aspri | Donkey milk: An overview on functionality, technology, and future prospects[END_REF]. Moreover, in the 15 DKM samples tested, no pathogens were detected above the limit prescribed by European and French regulations (EC 853/2004; Regulations of July 13, 2012): absence of Salmonella spp., absence of L. monocytogenes, less than 1 cfu/mL of Escherichia coli, and less than 10 cfu/mL of coagulase-positive staphy-lococci. The number of thermotolerant coliforms were 2.00 ± 1.50 log 10 cfu/mL, and the mean number of lactic acid bacteria were 2.17 ± 1.44 log 10 cfu/mL, in agreement with previous literature [START_REF] Chiavari | Use of donkey's milk for a fermented beverage with lactobacilli[END_REF][START_REF] Zhang | The antimicrobial activity of donkey milk and its microflora changes during storage[END_REF][START_REF] Šarić | Antibacterial properties of domestic Balkan donkeys' milk[END_REF][START_REF] Carminati | Biotyping of cultivable lactic acid bacteria isolated from donkey milk[END_REF]. These data show that French DKM is comparable to Italian, Chinese, or Balkan DKM, with good sanitary quality (low microbial load and absence of pathogenic microorganism); they illustrate good management and hygiene practices in French farms and should encourage French farmers to produce milk for human consumption.

Antimicrobial peptides such as lysozyme and lactoferrin may be responsible for the low bacterial load of DKM and can exert a positive effect on raw milk storage [START_REF] Zhang | The antimicrobial activity of donkey milk and its microflora changes during storage[END_REF]. The average lysozyme levels and LZA in the 15 farm samples tested were 2.74 ± 0.88 mg/mL and 17900 ± 1636 U/mL, respectively. Although the lysozyme levels mainly depend on the lactation stage and the production season, these concentrations are close to those described in Italian DKM [START_REF] Vincenzetti | Donkey's milk protein fractions characterization[END_REF][START_REF] D'alessandro | Major whey proteins in donkey's milk: Effect of season and lactation stage. Implications for potential dietary interventions in human diseases[END_REF]. The month of lactation affects the concentration of lysozyme in DKM, resulting in a higher concentration of lysozyme during the first 4 mo of lactation and a reduction thereafter [START_REF] D'alessandro | Major whey proteins in donkey's milk: Effect of season and lactation stage. Implications for potential dietary interventions in human diseases[END_REF]. Similarly, DKM whey protein concentrations are significantly higher at the beginning of lactation and decrease with time, as observed in humans [START_REF] Lönnerdal | Nutritional and physiologic significance of human milk proteins[END_REF][START_REF] D'alessandro | Major whey proteins in donkey's milk: Effect of season and lactation stage. Implications for potential dietary interventions in human diseases[END_REF]. Concerning LZA, linked to the different published methods of analysis used, the results obtained herein cannot easily be compared with those described in literature. To our knowledge, only a few studies have previously used the EnzCheck (Molecular Probes) assay, and these show similar LZA levels (15000 U/mL) to those obtained in our study [START_REF] Addo | Evaluating the ultra-high pressure homogenization (UHPH) and pasteurization effects on the quality and shelf life of donkey milk[END_REF]. Interestingly, LZA levels measured herein for DKM are close to those observed in human breast milk (18,000 U/mL; [START_REF] Sousa | Effect of thermal pasteurisation and high-pressure processing on immunoglobulin content and lysozyme and lactoperoxidase activity in human colostrum[END_REF].

No correlation between the enzymatic activity and levels of lysozyme was observed in our study (Figure 2), which suggests that a large quantity of lysozyme with low enzymatic activity is present in DKM. Due to their role in the development of infants, the amount of bioactive proteins in mammals' milk is considered important [START_REF] Butte | Human milk intake and growth in exclusively breast-fed infants[END_REF][START_REF] Lönnerdal | Nutritional and physiologic significance of human milk proteins[END_REF]. In newborns, the major function of lysozyme is to strengthen their immature immune system [START_REF] Cieslak | Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression[END_REF]. The biological properties of lysozyme are supported by 2 distinct mechanisms of action on bacterial structures, one of which involves the enzymatic activity of lysozyme, and the second consisting of a non-enzymatic mode of action via cationic properties, which results in a perturbation of the bacterial cyto-plasmic membrane [START_REF] Masschalck | Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure[END_REF]. In recent work, we have shown that daily consumption of DKM has anti-inflammatory properties in the gut, in a model of ileitis in mice [START_REF] Yvon | Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice[END_REF]. We observed that this dietary intervention requires significant enzymatic activity of lysozyme as a key condition to obtain a beneficial effect on gastrointestinal mucosa [START_REF] Yvon | Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice[END_REF]. Thermal treatments can reduce the nutritional quality of milk, affecting both the molecular structure and the enzymatic activity of milk proteins such as lysozyme. Based on these observations, we consider LZA to be a biologically active marker in DKM and focused our experiments on developing a heat treatment aimed at safeguarding the antimicrobial enzymatic activity of lysozyme. To define and optimize this heat treatment, we selected 1 DKM from La Ferme du Hitton, which combined good breeding and high quality of life for animals.

Effect of Thermal Treatments on Microbiological Quality and Enzymatic Lysozyme Activity

Although lysozyme is a temperature-resistant enzyme, heat treatments can significantly reduce its biological activity, with reductions ranging from 21 to 74% [START_REF] Wills | Short-time low-temperature pasteurisation of human milk[END_REF][START_REF] Koenig | Immunologic factors in human milk: The effects of gestational age and pasteurization[END_REF][START_REF] Czank | Simultaneous pasteurization and homogenization of human milk by combining heat and ultrasound: Effect on milk quality[END_REF][START_REF] Sousa | Effect of thermal pasteurisation and high-pressure processing on immunoglobulin content and lysozyme and lactoperoxidase activity in human colostrum[END_REF]. In our experiments, thermal treatments significantly (P < 0.05) affected LZA. (Figure 3A). The most drastic decreases were observed in treatments of 8 min at 72°C and 10 min at 80°C, which showed reductions of 27.15 ± 5.32% and 60.79 ± 11.65% of LZA, respectively, compared with LZA measured in raw DKM (Figure 3A). Heat treatment of DKM for 4 min at 72°C induced a reduction of 13.77 ± 2.70% of LZA compared with raw DKM. Pasteurization of DKM for 30 s, 2 min, or 3 min at 72°C had no significant effect (P < 0.05) on LZA compared with LZA measured in raw DKM (Figure 3A). Previous studies have showed that pasteurization in the same range of treatment times and temperatures had little effect on the content of other interesting compounds in DKM, such as immunoglobulins (IgA, IgG;[START_REF] Aspri | Donkey milk: An overview on functionality, technology, and future prospects[END_REF]vitamins (MacDonald et al., 2011) compared with raw DKM.

In the raw DKM tested, the total bacterial load was 3.90 log 10 cfu/mL. Thermal treatments safeguarding LZA-that is, 30 s at 72°C; 2 min at 72°C; or 3 min at 72°C-greatly decreased the total bacterial count (TBC), with a reduction of 2 log 10 cfu/mL, representing less than 100 cfu/mL in heated DKM (Figure 3B). Because our selected DKM contained a low load of total bacterial and no pathogen strains, to mimic poorer conditions of breeding and milking, 2 types of DKM were produced: DKM enriched with endogenous bacteria (Figure 3B) and DKM artificially contaminated with the thermotolerant E. faecalis (Figure 3B). Treatment for 30 s at 72°C reduced TBC by more than 4 log 10 cfu/mL (from 6.85 to 2.53 log 10 cfu/mL) in the bacteria-enriched DKM but had little effect (reduction of less than 2 cfu/mL of E. faecalis) on the artificially contaminated milk (from 7.56 to 6.11 log 10 cfu/mL). The 2 min, 72°C, treatment permitted a 2 log 10 cfu/mL reduction of TBC in enriched DKM and a 4.2 log 10 cfu/ mL reduction of E. faecalis in artificially contaminated DKM. Considering the low bacterial load in DKM (Table 3), we selected the 2-min treatment at 72°C, because it permitted reduction in both TBC and thermotolerant E. faecalis, while preserving LZA in DKM.

Aging Tests for Safety and Freshness Guarantees of Selected Heat-Treated DKM

According to the safety and hygiene criteria defined by the regulation of food microbiology no. 1441/2007and 852/2004(European Commission, 2004, 2007), samples of DKM previously submitted to heat treatment (2 min at 72°C) were analyzed at different time points. At 20 d post-treatment, total mesophilic bacteria were less than 10,000 cfu/mL. After 7, 10, 13, 15, and 20 d post-treatment, no L. monocytogenes and no salmonella were detected in DKM. For suspected enterobacteria, Escherichia coli-positive β-glucuronidase, and coagulase-positive staphylococci, fewer than 10 cfu/mL were counted, reflecting applicable criteria for DKM intended for human consumption. Indeed, an adequate heat treatment of milk must (1) make milk and milk products safe for human consumption by reducing the number of viable bacteria that may be harmful to health and (2) improve the shelf life of milk by reducing the numbers of spoilage bacteria [START_REF] Lejeune | Unpasteurized milk: A continued public health threat[END_REF]. In our study, the heat treatment applied (2 min at 72°C) extends the previous shelf life period described, 3 d at 4°C for raw DKM [START_REF] Zhang | The antimicrobial activity of donkey milk and its microflora changes during storage[END_REF], to 20 d at 4°C for treated DKM. Moreover, to further increase shelf life, milk can be frozen after pasteurization. Indeed, pasteurization and freezing after pasteurization are usual procedures carried out for human milk banks. Human breast frozen after pasteurization does not present any significant changes of the protein fraction [START_REF] García-Lara | Effect of Holder pasteurization and frozen storage on macronutrients and energy content of breast milk[END_REF]. Pasteurization before freezing also denaturizes enzymes involved in producing bitter flavor and rancidity of milk during storage [START_REF] Lemieux | Bitter flavour in dairy products. I. A review of the factors likely to influence its development, mainly in cheese manufacture[END_REF] and so should improve the taste and flavor of DKM.

Evaluation of Beneficial Effects of Raw vs. Heat-Treated DKM

In physiological conditions, oral administration of raw or heat-treated DKM (2 min at 72°C), containing similar LZA (5900 U), at 0.2 mL/d did not change basal total gut permeability in unstressed mice compared with unstressed control animals who received PBS [4.19 ± 0.68 and 4.04 ± 0.45, vs. 2.70 ± 0.28 ng of FITC per mL of plasma, respectively (Figure 4A); the obtained values were normalized by the BW (g) of each mouse]. In the same way, both treatments (raw or heat-treated DKM) had no effect on levels of the fecal inflammatory marker LCN-2 compared with control mice (23.57 ± 6.48 and 13.80 ± 3.09, vs. 15.75 ± 2.91 ng/g of feces, respectively; Figure 3B). In contrast, chronic psychological stress (WAS) induced a significant increase (P < 0.001) of total gut permeability to free FITC molecules (10.85 ± 1.06 ng/mL). This increased gut permeability is associated with a significant increase (P < 0.05) of LCN-2 levels (+84.48 ± 36.51 ng/g of feces) assessed in WAS mice. These effects of increased gut permeability and micro-inflammation were significantly counteracted (P < 0.01) by oral treatment with both raw and heat-treated DKM. To reinforce these data, further experiments using several milk samples from different farms and countries should be performed, to consolidate their effects on gut function after this specific heat treatment. As described in the introduction, DKM is a complex matrix containing several mediators such as interferon γ (IFNγ), lysozyme, lactoferrin, and TGF-β. Therefore, in our study, we cannot exclude the possibility that heat-treated DKM could exert beneficial effects on gut functions not only via lysozyme activity but also via other mediators that might resist the treatment process.

Increased intestinal epithelial permeability has been implicated as a prerequisite in the pathophysiology of IBS [START_REF] Spiller | Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome[END_REF] and has also been found in animal models of chronic stress [START_REF] Söderholm | Chronic stress induces mast cell-dependent bacterial adherence and initiates mucosal inflammation in rat intestine[END_REF][START_REF] Barreau | 0.05; ***P < 0.001 compared with stressed mice. #P < 0.05; ##P < 0.01 compared with consumption of water among stressed mice. Gray shaded bars = unstressed mice (control); black bars = mice subjected to water-avoidance stress. P = pasteurized. Error bars represent standard error of the mean. mucosal barrier induced by neonatal stress in rats[END_REF]. Additionally, IBS is associated with mucosal immune stimulation, a micro-inflammatory state characterized by increased density of immunocytes and mast cells in the gut mucosa [START_REF] Guilarte | Diarrhoea-predominant IBS patients show mast cell activation and hyperplasia in the jejunum[END_REF]. Altered mucosal barrier permeability facilitates the entry of toxins and bacteria and is considered to be responsible for mucosal micro-inflammation [START_REF] Hyland | Microbiota-host interactions in irritable bowel syndrome: Epithelial barrier, immune regulation and brain-gut interactions[END_REF]. Furthermore, in IBS, increased gut permeability is positively correlated with visceral pain [START_REF] Zhou | Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome[END_REF], and in rodents stress-induced gut hyperpermeability is also responsible for visceral hypersensitivity [START_REF] Ait-Belgnaoui | Acute stress-induced hypersensitivity to colonic distension depends upon increase in paracellular permeability: role of myosin light chain kinase[END_REF]. In this context, alteration of intestinal permeability constitutes a clear target in pathophysiological treatment for IBS today.

CONCLUSIONS

Based on the beneficial effect of DKM, heat treated according to our defined procedure, on WAS-induced intestinal damage and the observations previously published in preclinical stress models and in human IBS patients, we propose oral consumption of DKM as an intervention against intestinal barrier dysfunction due to psychological stress. However, other studies are needed to assess the efficacy and safety of DKM in the management of IBS symptoms. We first suggest testing the beneficial effects of DKM on a model of stress-induced visceral hypersensitivity.

  Pasteurized DKM. An aging test of the selected pasteurized DKM (2 min/72°C) was performed at Lanagram Laboratories (Mazamet, France). The health and safety criteria were determined in 100 g of DKM samples obtained at 1, 7, 10, 15, and 20 d post-heat treatment. More precisely, counts (cfu) from total microorganisms (NF EN ISO 4833-1), enterobacteria (NF V 08-054), B-glucuronidase-positive Escherichia coli (NF ISO 16649-2), coagulase-positive staphylococci (NF EN ISO 6888-2), and the presence or absence of Salmonella spp. (AES 10/11-07/11) and L. monocytogenes (BKR 23/2-11/02) were monitored.

Figure 1 .

 1 Figure 1. Experimental design of the study. (1) Diagnosis phase; evaluation of the health quality of raw donkey milk (DKM) from 15 French farms. (2) Optimization of a thermization process to preserve lysozyme activity and reduce harmful microorganisms. (3) Evaluation of beneficial effects of thermized DKM on altered gut barrier in mice exposed to water-avoidance stress (WAS).

Figure 2 .

 2 Figure 2. Correlation between enzymatic activity (U/mL) and concentration of lysozyme (mg/mL) in donkey milk from 15 French farms. Linear regression: R 2 = 0.000784, indicating that the equation of the regression line determines 0.0784% of the distribution of points.

Figure 3 .

 3 Figure 3. (A) Effects of thermal treatments (pasteurization, P) on enzymatic lysozyme activity in bacteria-enriched raw donkey milk (DKM): *P < 0.05, **P < 0.01, compared with non-heat treated (NT) raw DKM. (B) Effects of thermal treatments on total bacterial count (TBC) in DKM: *P < 0.05, **P < 0.01 compared with NT (donkey milk, unshaded bars); ++P < 0.01 compared with bacteria-enriched DKM (donkey milk + TBC, gray-shaded bars); and #P < 0.05, ##P < 0.01 compared with DKM contaminated with Enterococcus faecalis (donkey milk + E. faecalis, black bars). Error bars represent standard error of the mean.

Table 1 .

 1 Methods used in microbiological analysis to evaluate the health quality of raw donkey milks selected from 15 French farms

			Incubation	Inoculation
	Groups of microorganisms	Medium	conditions	method
	Total bacterial count (TBC)	Plate count agar (PCA) + 0.1% skim milk powder (Biokar 1 )	30°C, 72 h	Inclusion (1 mL)
	Thermotolerant coliforms	Violet red bile lactose (Biokar)	44°C, 48 h	Inclusion (1 mL)
	Lactic acid bacteria	Elliker modified by Chamba et al. (1981) pourpre de bromocrésol (Acros Organics 2 ) + thallium acetate (Sigma-Aldrich 3 )	30°C, 72 h	Surface (100 µL)
	Total staphylococci	Columbia blood agar (Sigma-Aldrich)	37°C, 48 h	Surface (100 µL)
	Enterococcus faecalis	Brain-heart infusion agar 15% (Biokar)	37°C, 48 h	Surface (100 µL)

Table 2 .

 2 Heat treatment conditions

	Treatment	Temperature (°C)	Time (min)
	1	72	0.5
	2	72	2
	3	72	3
	4	72	4
	5	72	8
	6	80

Table 3 .

 3 Microbiological characteristics and lysozyme content and activity of French raw donkey milk; values ± standard deviation

	Item	Value
	Bacterial count (log 10 cfu/mL)
	Total mesophilic bacteria	3.82 ± 1.56
	Lactic acid bacteria	2.17 ± 1.44
	Thermotolerant coliforms	2.00 ± 1.50
	Staphylococci (coagulase positive)	<10 cfu/mL
	Escherichia coli	<1 cfu/mL
	Salmonella spp. in 10 g	0
	Listeria monocytogenes in 10 g	0
	Lysozyme
	Level (mg/mL)	2.74 ± 0.88
	Activity (U/mL)	17,900 ± 1,636

Journal of Dairy Science Vol. 102 No. 9, 2019

ACKNOWLEDGMENTS

The authors thank Christel Couderc, Mireille Gaucher, and Henri Tonglet (Institut National Polytechnique-Purpan, Toulouse, France) for their technical assistance. The authors also thank the Institut National Âne et Mulet (INAM) and the Ecole d'Ingénieurs de Purpan for their financial support. Thanks to Marlene Fraysse (Valence d'Agen, France) and Jasper Kamphuis (Institut National de la Recherche Agronomique Toxalim, Toulouse, France) for their proofreading.