Julie Bourbeillon

Catherine Garbay

Joëlle Simony-Lafontaine

Françoise Giroud

Multimedia Data Management To Assist Tissue Microarrays Design

Keywords:

Introduction

Tissue Microarray (TMA) technology [START_REF] Kallioniemi | Tissue microarray technology for high-throughput molecular profiling of cancer[END_REF] is a new technique used in oncology research. It allows for quick in situ visualisation of molecular targets in thousands of tissue samples. In this technique patients are selected according to the study to be conducted. A pathologist analyses a histological slide for each patient biopsy block and marks areas of interest. Tissue cores are extracted from each biopsy paraffin block (donor block) and inserted in a new paraffin block (receiver or TMA block) in which slides are cut and treated as conventional slides would be. Images for TMA slides are acquired annotated and submitted to image analysis treatments.

Compared to classical studies, those using TMA technology economise on reactants and biological material. Moreover, mass treatment brings a statistical dimension to the pathologist's work. Both advantages can be increased by using the virtual TMA slide concept: spot images can be reorganised according to a new study without constructing a new real block. However TMA technology suffers from a lack of formalised knowledge and automation of TMA design and data analysis.

A major step in the computerisation of TMA technology has been the definition of the TMA Data Exchange Specification [START_REF] Berman | The tissue microarray data exchange specification: a community-based, open source tool for sharing tissue microarray data[END_REF]. But most tools focus on data management [START_REF] Henshall | Tissue microarrays[END_REF], and an increasing need for assistance in data mining is being noticed [START_REF] Shergill | Tissue microarrays: a current medical research tool[END_REF]. We therefore intend to set up an integrated web-based system providing assistance in real TMA block design and TMA data mining through the generation of virtual representations of future TMA blocks or virtual TMA slides. The system will have to generate these representations "on the fly", according to a user query. These will have to be a complex mix of heterogeneous data adapted to users' needs.

In this paper we present preliminary work to formalise this adaptation process.

Related Works

User-adaptive software systems and especially adaptive hypermedia and adaptive web [START_REF] Brusilovsky | From Adaptive Hypermedia to the Adaptive Web[END_REF] compute the layout and contents of web pages according to a user model. Beyond this simple personalisation according to preferences, some systems such as e-Learning or on-line newspapers [START_REF] Iksal | Adaptive Special Reports for On-line NewsPapers[END_REF] aim at representing a narrative coherence through a spatial organisation. Compared to such approaches the envisioned system intends to reach a representative thematic organisation according to a query which is analytic by nature. This implies that we also focus on task adaptation.

Indeed, as with all information retrieval systems the goal is to provide some relevant information according to a user query. In our case however, as with web page summarising [START_REF] Mckeown | Tracking and Summarizing News on a Daily Basis with Columbia's Newsblaster[END_REF] or news tracking systems (such as News-map [8]), the returned data must be seen as an organised collection of documents. In the pictured system, the collected data is even meant to support data mining operations. As a consequence, the retrieved data has to be selected and organised to present an overview of the complete collection through a representative set which keeps the variety present in the original collection and allows for statistical analysis.

To achieve these adaptations, it is necessary to acquire and represent some knowledge about the adaptation task and the application domain. Using ontologies [START_REF] Gruber | Toward principles for the design of ontologies used for knowledge sharing[END_REF] will facilitate the modelling and knowledge sharing amongst human and software agents.

TMA Design: Problem Overview

Given a request the document to be designed may be thought of as an arrangement of objects, each being characterised by information extracted from a data repository. Therefore virtual representations of real TMA blocks and virtual TMA slides can be described as collections of multimedia documents that are generated according to a user request. They consist of a user query defining the study that the biologist or the physician wants to conduct (for instance comparing two patients groups...) and a TMA grid (the assembly of spot images selected and spatially arranged according to the user query). Each spot can be linked with data regarding the corresponding patient (clinical data...), image annotation and analysis (staining quantification...).

Starting from available data and knowledge, the tool we intend to build has to generate the TMA multimedia document on the fly. This is a complex problem which can be split into three sub-problems:

1 Selection: Building a TMA can be thought of as looking at a list of elements (biopsy areas or spots) for a collection (the list for pertinent patients or spots) which fits the demand (the user query) and complies with some general rules. 1 Spatial organisation: These objects (cores or spots) have to be placed on a grid. 1 Presentation: The previously generated set has to be displayed to the user in the most user-friendly way while still allowing for varying user preferences. The complexity of the problem is first of all linked to the heterogeneity of the data to handle. Some elements are purely computer data (virtual objects such as the numeric representation of the slides) and others are references to real objects such as the slides themselves. Some, such as staining intensity, are quantitative, whereas oth-ers such as patient gender are qualitative. The difficulty is increased due to the associated combinatorics.

Moreover the query is more than just inclusion/exclusion criteria since the aim is to obtain a spatially organised collection of documents which will be used for data mining. As a consequence, it appears necessary to acquire some knowledge about the TMA technology domain and the adaptation process at hand. This will be achieved using ontologies.

TMA Design: Ontologies

Building a TMA design ontology first of all requires modelling the pathology field. Currently available medical ontologies are either too general for our purpose or do not include abnormal tissue structures [START_REF] Rosse | A reference ontology for biomedical informatics: the Foundational Model of Anatomy[END_REF]. As a consequence, a colon carcinoma ontology has been put forward by Dr. Joëlle Simony-Lafontaine (a pathologist from the Centre Régional de Lutte Contre le Cancer in Montpellier, France). #A3C6B432AA34 9A9ABA23DA34!$A5B3C235425AB3CBB45CA3D3DA3A5D2464783663DABA32442B C9A32A79CA323C3F34C234246478

Along this domain ontology a task ontology needs to be modelled. An example of a query made by a pathologist expressed in natural language might be: 'Study of colon cancer evolution among men with a virtual TMA slide and using the Ki67 marker.' Such a query can be decomposed into several elements. The Goals of the Study guide the way to arrange the objects into the TMA grid. Inclusion Criteria define the list of patients or spots to take into account to build the grid. The Samples are a list of interesting areas in the biopsies where samples should be taken. The Document Logical Model is the TMA document type to build. The Lab protocols list specific protocols to build the TMA. This decomposition led us to construct a query ontology which guided the formalisation presented in Table 1.

The generation of a TMA document is a process of selection, spatial organisation on a grid, and presentation of this grid to the user. This is a process of adaptation of the final document to a query which is directed by a set of criteria. Some of these criteria are specific to the application domain, whereas others are specific to the query, and they can be influenced by user preferences expressed in the query or in a profile.

Query Element Problem Element

Formalism ( [Father Element]...) [Element] ( = [Value]) Goal of the Study Organisation

[Goal] [Patient] [Diagnostic] [pTNM grade] Inclusion Criteria Selection [Inclusion Criteria] [Patient] [Sex] = [Male] Samples Selection [Sample] [Area] = [Colon] [All] Document Logical Model Selection / Organisation [Document Logical Model] = [Virtual] Lab Protocols Selection / Organisation [Lab Protocol] [Grid Size] = [Default] [Lab Protocol] [Nail diameter] = [Default] [Lab Protocol] [Marker] = [Ki67]
Given the collection of criteria introduced in the previous section the TMA design issue consists of selecting and ordering a group of relevant criteria, and categorising them according to the query and user preferences in order to propose an adaptation plan. The manipulation of such a criteria collection is a complex problem because of its size and possible contradictions. However the studies that can be conducted can be classified into families of similar studies. We can also build an adaptation model for each of these families. An adaptation model consists of a more specialised subset of constraints and rules extracted from the whole collection according to the specifics of the corresponding study family.

To achieve the adaptation of the final TMA document to the task those adaptation models have to be instantiated with data extracted from the query. As a consequence adaptation models constitute a representation layer depending on the goal of the study which plays an intermediate role between the domain and query layers.

Three specialisation layers have then been defined. The Domain Layer includes the complete collection of criteria for the considered application domain. The Goal Layer includes a subset of the previous collection, selected, ordered and parametrised according to the goal of the corresponding query family. The Query Layer includes an adaptation model specialisation according to the current query and user preferences.

Following these three specialisation layers, three composition levels corresponding to the three sub-problems to solve can be defined. The Factual Level deals with the selection stage where data or facts are analysed in order to present a pertinent element list. The Logical Level deals with the spatial organisation step where a thematic ordering of the previous list is achieved. The Lay-out Level deals with the presentation stage where the the previous document is prepared to be displayed while taking into account some user preferences.

A TMA document generation is a two-stage process which is achieved by the adaptation engine presented Figure 1. The adaptation engine goes through the three specialisation layers to generate the Adaptation Plan. The application of the adaptation plan to the three composition levels allows to create the final TMA document.

Conclusion

In paper we have proposed an architecture for an adaptation engine which for now, is dedicated to TMA design assistance. The proposed architecture relies on an adaptation engine operating along two axes. The multi-layer Specialisation consists of a progressive refinement of the adaptation procedure towards a particular case. It facilitates knowledge representation and acquisition and allows for flexible query formulation. The multi-level Composition consists of a progressive refinement of the adaptation process through successive steps. It allows for task decomposition and it eases the formalisation of expertise.

The next step would be to go further into the conception and build a prototype.

Fig. 1 .

 1 Fig. 1. Adaptation Engine Architecture

Table 1 .

 1 Example for a formalised query where the elements are referenced according to the query ontology.


Spheroid Intissar Razgallah
email: intissar.razgallah@utc.fr

Rafik Ouchene

Mohamed Khalij
email: mohammed.khalij@univ-lorraine.fr

Borice Arcen

Aanne Taniere

NUMERICAL INVESTIGATION OF HEAT TRANSFER AND FLUID FLOW BEHAVIOR AROUND AN OBLATE

Keywords: Convection, heat transfer, oblate spheroid, sphere, aspect ratio, orientation, Nusselt number, drag coefficient, angle of attack

In this study, a numerical approach was adopted to investigate the heat transfer of an oblate spheroid in a steady flow of viscous and incompressible fluid characterized by Prandtl Numbers with values of 0.7, 1 and 7. The temperature of the spheroid is considered constant. This hypothesis is valid as the thermal conductivity of the particle is supposed to be much greater than the conductivity of the fluid. The uncoupled momentum and heat balance equations were solved numerically for particular Reynolds Numbers ranging from 0.1 to 100, corresponding to a laminar axisymmetric regime. Case studies were done on particles with aspect ratios of 0.2, 0.5 0.8 and 1. The validation of this work included a comparison of the numerical results of the drag coefficient, the local and the average Nusselt Numbers, to results found in literature and by visualization of the flow around the particle. The computations were focused on the influence of the axis ratios and the angle of attack on the heat transfer rate. It, also, shows the influence of the choice of the reference length of the spheroid, considered in the actual paper equal to the equivalent diameter of the sphere, on the variation of the Nusselt Numbers.

Introduction

Because of the inaccuracy produced by the sphericity assumption that was adopted in chemical engineering, scientists are keen to find more accurate shape assumptions to provide more reliable results in describing the flow and heat transfer around particles in industrial processes. These processes include combustion, chemical reactions, mixing and separation processes. Among these applications, oblate spheroidal shape seems to more precisely approximate the geometries in question and represent a good approximation for non-spherical particles [START_REF] Clift | Drops and Particles[END_REF]. Pulverized coal is for instance a good example to illustrate the products encountered [START_REF] Richter | New correlations for heat and fluid flow past ellipsoidal and cubic[END_REF][START_REF] Ouchène | Drag lift and torque coefficients of the ellipsoidal particles[END_REF]. In order to insure a better efficiency, it is of high interest to control with great accuracy such industrial processes through the knowledge of the settling velocity, which is greatly related to the drag forces, and the heat transfer coefficient usually determined by the Nusselt Number. A number of research papers have already treated the subject. However, limited attention was given to the effect of the emerged particle orientation on flow and heat transfer. Therefore, this will be the aim of this study. Thus, we express the orientation of the particle in terms of an angle of attack. Defined as the angle between the particle axis of revolution and the direction of flow, this angle was varied between 0° and 90° degrees.

Overview of the literature

We start this work by presenting an insight of the literature on unconfined spheroidal particles. Motion in creeping flow was first investigated by Stokes [START_REF] Stokes | On the effect of the inertial friction of fluids on the motion of pendulums[END_REF] on spherical particles. He developed the well-known expression for the drag coefficient by neglecting the inertial terms. Using the same approach, it was Oberbeeck [START_REF] Oberbeek | Ueber stationäre Flüssigkeitsbewegungen mit Berücksichtigung[END_REF], who started a similar work concerning ellipsoidal particles. Based on his results, Happel and Brenner [START_REF] Happel | Low Reynolds Number Hydrodynamics[END_REF] developed an expression for the drag coefficient by introducing an additional shape correction to the Stokes drag coefficient. Those shape correction factors, developed using an asymptotic method, also take the particle orientation into account. By determining the drag coefficient for the extreme angles of attack of 0° and 90°, a function linking these two limiting cases, with an inflection point at 45, can be used to represent the whole range of incidence angles for a non-spherical particle. As for spherical particles, the orientation has no effect on the motion of the particle. Therefore, the shape correction is constant and equal to 1.

For higher Reynolds Numbers a number of experimental and numerical studies were carried out in order to describe the drag coefficient in terms of Reynolds Numbers and the particle orientation. For the case of an ellipsoidal particle with an aspect ratio of 1, the particle is reduced to a sphere. The experimental study conducted by Schiller and Naumann [START_REF] Schiller | A drag coefficient correlation[END_REF] seems the most widely used correlation in this case. On the other hand, for an ellipsoidal particle most of the studies carried out are based on a numerical approach. Militzer et al. [START_REF] Militzer | Drag coefficient for axisymmetric flow around individual sphéroidal particles[END_REF] used the existing data to develop a correlation describing the drag coefficient of a spheroid by introducing the aspect ratio into the Schiller and Naumann correlation [START_REF] Schiller | A drag coefficient correlation[END_REF]. The expression obtained is useful for particles of aspect ratios ranging between 0.2 and 5 and for Reynolds Numbers under 200. Unfortunately, this correlation doesn't take into account the orientation of the particle.

( )         - - - - - = = - 2 1 2 3 2 2 2 3 1 1 sin 1 3 2 1 3 8 ) 0 ( E E E E E E K  ( )         - - - - = = - - E E E E E E E K 1 tan 1 ) 2 1 ( 2 1 2 3 8 ) 90 ( 2 1 2 3 2 2 2 3 1



Other studies succeeded in taking the orientation of the particle into account. The most important ones are those conducted by Rosendhal [START_REF] Rosendahl | Using a multi-parameter particle shape description to predict the motion of non-spherical particle shapes in swirling flow[END_REF], Zastawny et al. [START_REF] Zastawny | Derivation of drag and lift force and torque coefficients for nonspherical particles in flows[END_REF], Ouchene et al. [START_REF] Ouchène | Drag lift and torque coefficients of the ellipsoidal particles[END_REF], and Hölzer and Summerfeld [START_REF] Hölzer | New simple correlation formula for the drag coefficient of non-spherical particles[END_REF]. The main difference between these correlations is the use of the parameters to describe the non-spherical shape of the particle and its orientation. Hölzer and Summerfeld used the sphericity and crosswise spherecity to characterize respectively these two parameters. Rosendhal, on the other hand, used first these two parameters to determine an expression of the drag coefficient at the extreme angles of attack. Then, he used a connecting function depending on the angle of attack. The rest of the studies were similar to Rosendhal's study except that they used the aspect ratio instead of the sphericity in establishing the correlations to describe the extreme values of the drag coefficient.

It was Fourrier [12], who first investigated a spherical particle placed in a surrounding fluid and gave an asymptotic expression for the Nusselt Number in a stagnant fluid. He proved that the diffusion is completely radial and results on a Nusselt Number equal to 2. More appropriate results in creeping flows were established by Brenner [START_REF] Brenner | Forced convection heat and mass transfer at small Péclet numbers from a particle of arbitrary shape[END_REF] who developed an analytical expression valid for small Peclet Numbers.

For infinite Peclet Numbers, Levich [START_REF] Levich | Physicochemical hydrodynamics[END_REF] used the thin boundary layer theory to develop an expression for the Nusselt Number that was enhanced later by Acrivos and Goddard [START_REF] Acrivos | Asymptotic expansions for laminar forced-convection heat and mass transfer[END_REF] using a small perturbation method. However, Friedlander [START_REF] Friedlander | Mass and heat transfer to single spheres and cylinders at low Reynolds numbers[END_REF] was able to find a better expression.

Fourrier [12] 2 = Nu Clift et al. [1] ( ) 3 1 Pr Re 1 1 + + = Nu Friedlander [16]             - = 2
      + = a a Pe Nu Nu Nu 2 0 0 2 8 1 1
Brenner second order development [START_REF] Brenner | Forced convection heat and mass transfer at small Péclet numbers from a particle of arbitrary shape[END_REF] Massliyah and Epstein [START_REF] Masliyah | Numerical solution of heat and mass transfer from spheroids in steady axi-symmetric flow[END_REF] 3 1

2 3 3 0 0 a 2 2 2 Nu         +       + = a Pe K Nu Nu
As for non-spherical particles, the diffusive heat transfer in a stagnant media was investigated by Smith, Weber and Schneider. Particularly, in the case of an oblate spheroid, the Nusselt Number expression depended on the orientation of the particle and was given for an angle of attack between 0° and 90°.

A valuable work was established in a Stokes flow regime by Brenner [START_REF] Brenner | Forced convection heat and mass transfer at small Péclet numbers from a particle of arbitrary shape[END_REF] using a matched asymptotic expansion method. The expression provided by him relies on the Nusselt Number in stagnant fluids and is adapted to arbitrary shapes. The orientation of the particle is taken into account by a dimensionless drag parameter defined as the ratio of the drag of an arbitrary shaped particle to the drag of the equivalent spherical particle.

Another expression by Sehlin [START_REF]Forced convection heat and mass transfer at large Peclet numbers from axisymmetric body in laminar flow: prolate and oblate spheroids[END_REF] for heat and mass transfer from a spheroid particle was derived for infinit Peclet Numbers using the thin boundary layer theory. The established correlation is similar to Levich's expression modified using a concentration factor. Beside these analytical results, numerical studies on creeping flow were conducted by Massliyah and Epstein [START_REF] Masliyah | Numerical solution of heat and mass transfer from spheroids in steady axi-symmetric flow[END_REF], Clift et al. [START_REF] Clift | Drops and Particles[END_REF] and Alassar [START_REF] Alassar | Forced convection past an oblate spheroid at low Reynolds numbers[END_REF].

For higher Reynolds Numbers, the heat transfer around a sphere was extensivly studied and a number of correlations were developed using expeimental and numerical approaches. The oldest are those established by Ranz and Marshall [START_REF] Ranz | Evaporation from drops[END_REF] and Whitaker [START_REF] Whitaker | Forced convection heat transfer correlations for flow in pipes, past flat plates, single cylinders, single spheres, and for flow in packed beds and tube bundles[END_REF]. Newer Correlations were then established by Clift et al. [START_REF] Clift | Drops and Particles[END_REF], Li and Mason [START_REF] Li | A computational investigation of transient heat transfer in pneumatic transport of granular particles[END_REF], and Feng and Michailides [START_REF] Feng | Heat and mass transfer coefficients of viscous sphere[END_REF] adapted for a wide range of Reynolds and Prandtl Numbers. 10≤𝑅𝑒 ≤ 200 Pr=0.74

In the case of ellipsoidal particles, few correlations had been established for particles with a flat orientation including the studies by Comer and Kleinstreuer [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF], by Clift et al. [START_REF] Clift | Drops and Particles[END_REF] and by Srinivas and Ramesh [START_REF] Srinivas | Numerical analysis of Heat Transfer from a Spheroidal Shaped Body to a Power Law Fluid at Finite Reynolds Number[END_REF]

+ = - - = 1 ≤𝑅𝑒 2𝑎 ≤ 100 0.2 ≤ E ≤5
Srinivas and Ramesh [START_REF] Srinivas | Numerical analysis of Heat Transfer from a Spheroidal Shaped Body to a Power Law Fluid at Finite Reynolds Number[END_REF] ( )

E Exp Nu Nu E E a 256 . 0 2917 . 1 1 1 , 2 - = =  0 ≤𝑅𝑒 2𝑎 ≤ 100 1≤ Pr ≤100 0.5 ≤ E ≤2 0.4 ≤ n ≤1.6
Richter and Nikrityuk [START_REF] Richter | New correlations for heat and fluid flow past ellipsoidal and cubic[END_REF] 𝑁𝑢 𝑑𝑒 = 1.59 + 0.64𝑃𝑟 These correlations are valid on a small range of Peclet Numbers. For a 90° angle the only correlations known were developed by Kishore and Gu [START_REF] Kishore | Momentum and heat transfer phenomena of spheroid particles at moderate Reynolds and[END_REF] and Sreenivasulu et al. [27] As the first correlation has a wide range of validity, it seemed less accurate at hight Prandtl Numbers compared to the correlation established by Sreenivasulu et al. with a maximum relative error reaching 28% between their numerical results and the correlation. On the other hand, the correlation by Sreenivasulu et al. seems to cover only Prandtl Numbers larger than 5.

Thus we need more accurate correlations for these extreme angles. Since there seems to be no published research that investigates the effect of intermediate angles on heat transfer we have run a numerical study to investigate this effect and try to find a correlation which takes this aspect into account.

Problem formulation and governing equations

In this work, a solid oblate spheroid of constant temperature Ts is immerged in a homogeneous unbounded Newtonian fluid. With constant properties, the velocity fluid field is independent of its temperature field. We consider that the conductivity of the particle is very important compared to the conductivity of the fluid which allow us to suppose an instant homogenization of the temperature of the particle on its surface. The fluid is supposed to have a uniform velocity U0 far from the particle. Its temperature is maintained constant T∞ and lower than Ts, with |Ts -T∞|=100K. Since, in this paper, we use only small Mach numbers, we can consider the fluid as being incompressible.

Its density is thus constant and we can neglect buoyancy effects. The object is of length '2a' in the direction normal to flow and '2b' in the direction parallel to flow called the major and minor axes respectively. Characterizing an oblate spheroidal shape, the aspect ratio is defined as the ratio of the ellipsoidal minor to major radius E = (b/a), ranges between 0 and 1 for an oblate spheroid. If E tends to 0, the particle considered is a flat circular disk. Whereas, if E=1 the particle is identical to a sphere. In this study, we will examine the cases for E=1, E=0.8, E=0.5 and E=0.2 (fig. 1).

Fig. 1 : Scheme of investigated particles

The heat transfer process is governed by the conservation equations.

𝑑𝑖𝑣 (𝑢 ⃗ 𝑓 ) = 0

(1)

g u p grad Dt u D f f f f    +  + - =   1 (2) 𝜕𝑇 𝑓 𝜕𝑡 = 𝑢 ⃗ 𝑓 𝑔𝑟𝑎𝑑 ⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ ( 𝑇 𝑓 ) + 𝐾 𝑓 ∆ 𝑇 𝑓 (3)
𝐷𝑇 * 𝐷𝑡 = 1 𝑅𝑒 𝑃𝑟 ∇ 2 𝑇 * (6)
Where Reynolds and Prandtl numbers are dimensionless groups deduced from the non-dimensional form of the equations. The boundary conditions are specified in fig. 2. The usual boundary conditions are the no slip conditions on the body and the free stream conditions far away from the body.

Simulation

Using FLUENT 15.0, the equations of conservation (eqn (1), eqn (2) and eqn (3)) were solved for a certain number of cases. The different cases simulated are given in table 1. For this purpose, we used the SIMPLE algorithm for Pressure-Velocity Coupling that suits unstructured meshes. The domain is chosen to have a cuboid form. It is schematized on fig. 2. Consisted of tetrahedral elements, our mesh is sufficiently refined near the surface of the particle using the method of gradual inflation and captures the dynamic boundary layer by a minimum of 40 nodes (fig. 3). As for spatial discretisation, it was done using a second order upwind scheme.

Validation of the simulation results

Before analysing the results of the present work it is important to make sure the correctness of the numerical scheme. First, we asserted that the dimensions of the domain are sufficiently large enough in order to insure that the fluid at the boundaries is not influenced by the walls. On the other hand, it should be as small as possible to reduce the calculation time. For this purpose, we tested three dimensions (table 2). Knowing that the boundary layer is thicker as the Reynolds number is smaller, the drag coefficient was compared for the different domain dimensions considering a Reynolds number equal 0.1. We notice that we get a sufficient result for a domain with a length equal 40 dp. It is also important to ensure a sufficient number of nodes in the area near the surface of the particle, where the velocity and temperature gradients are the greatest. For this purpose we compared the results obtained for average Nusselt number and drag coefficient obtained for three grids with different nodal numbers. In table 3, we notice that we receive sufficient results with grid A. There is only an error smaller than 1% between Grid A and Grid C if we compare the Nusselt Number obtained for each case. So we fixed the number of nodes for all the simulated cases to the same number imposed by grid A.

Comparison in the case of the sphere

In this section we discuss the different results obtained during the simulation and we compare them to the results found in the literature. A comparison in table 4 of the drag coefficient obtained by simulation and the results by Stokes and by Schiller and Naumann [START_REF] Schiller | A drag coefficient correlation[END_REF] was possible for a Reynolds of 0.1. It shows a relative error of 1% between our results and those by Schiller and Naumann [START_REF] Schiller | A drag coefficient correlation[END_REF] and a 3% of relative error between our results and those by Stokes [4]. This is mostly due to the fact that the relation by Stokes was neglecting the friction at small Reynolds numbers. For higher Reynolds numbers, the comparison was only possible for the results from the equation of Schiller and Naumann [START_REF] Schiller | A drag coefficient correlation[END_REF] (Fig. 4). Here, we obtained again a good accuracy with 4% as a maximum error, and an average relative error of 1.9%.

Fig.4 : Comparison of drag coefficient for high Reynolds numbers

The average Nusselt number is defined in spherical coordinates as shown in eqn [START_REF] Schiller | A drag coefficient correlation[END_REF], where  Nu is the local Nusselt number and Nu, is the average Nusselt number. The calculations were done with an equivalent diameter equal to the sphere diameter. [START_REF] Schiller | A drag coefficient correlation[END_REF] The local Nusselt number is defined by eqn( 8)

        -   - =    T T n T Nu s paroi f (8)
In order to verify the results obtained for heat transfer, we chose to compare the results of the Nusselt average obtained numerically, for Reynolds at 0.1 in a creeping flow and different Prandtl numbers, to results from Fourrier [12], Clift et al. [START_REF] Clift | Drops and Particles[END_REF] and Friedlander [START_REF] Friedlander | Mass and heat transfer to single spheres and cylinders at low Reynolds numbers[END_REF]. As shown in table 5, our numerical results are close to the asymptotic solution by Fourier [12]. The relative error between our results and those of Friedlander increases up as the Peclet number become larger than 0.1. In that case, the conduction is dominating. The correlation by Clift et al. [START_REF] Clift | Drops and Particles[END_REF] shows smallest error to ours with a maximal relative error of 1.7%. Since the convective heat transfer coefficient varies over the surface of the sphere, we expect the Nusselt number to vary locally. This variation is mainly influenced by the flow regime of the fluid that surrounds it. In Fig. 6, the variation of local Nusselt number is plotted dependent on the polar angle expressed here in Radian for a Reynolds of 0.1, and for the different values of the Prandt number studied. The local Nusselt number increases as the number of Prandtl becomes larger. This is due to the fact that the convective heat transfer rises as Prandtl increases. Moreover, we note that the curve have a maximum at the breakpoint level. Following which the local Nusselt number decreases gradually as one moves away from the stop point downstream of the obstacle. This decrease is due to the fact that the boundary layer becomes less dense. For high and intermediate Reynolds numbers, a comparison of our numerical results was possible with the results by Feng and Michailides [START_REF] Feng | Heat and mass transfer coefficients of viscous sphere[END_REF], Clift et al. [START_REF] Clift | Drops and Particles[END_REF], Comer and Kleinstreuer [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF], Li and Mason [START_REF] Li | A computational investigation of transient heat transfer in pneumatic transport of granular particles[END_REF], Whitaker [START_REF] Whitaker | Forced convection heat transfer correlations for flow in pipes, past flat plates, single cylinders, single spheres, and for flow in packed beds and tube bundles[END_REF] and Richter and Nikrityuk [START_REF] Richter | New correlations for heat and fluid flow past ellipsoidal and cubic[END_REF] (fig. 7). For all the Prandtl numbers considered, excellent compatibility was found between our simulation results and the correlation established by Feng and Michailides [START_REF] Feng | Heat and mass transfer coefficients of viscous sphere[END_REF] as well as those established by Clift et al. [START_REF] Clift | Drops and Particles[END_REF] (table 6). However, the latter appears to underestimate the results for Pr = 7 when the Reynolds number is greater than 60 with a maximum relative deviation equal to 4.4%. The correlations of Comer and Kleinstreuer [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF] for a Prandtl numbers of 7 and 0.7, seems to describe with good accuracy the average Nusselt number for a Reynolds number greater than 10. As for the correlations of Li and Mason [START_REF] Li | A computational investigation of transient heat transfer in pneumatic transport of granular particles[END_REF] and Whitaker [START_REF] Whitaker | Forced convection heat transfer correlations for flow in pipes, past flat plates, single cylinders, single spheres, and for flow in packed beds and tube bundles[END_REF], they seem to underestimate or overestimate the average Nusselt number especially for high Reynolds number. For a Prandtl number of 0.7, the correlation by Richter and Nikrityuk [START_REF] Richter | New correlations for heat and fluid flow past ellipsoidal and cubic[END_REF] seems best suited with a maximum relative error of 1.4% regardless of the Reynolds considered between 1 and 100. At large Prandtl numbers, strong convective currents yielded increased Nusselt numbers.

As for the local Nusselt number, its evolution in terms of the polar angle was schematized in fig. 8 for each Reynolds number and Prandtl number studied. The first thing that comes to mind is the increase of local heat transfer when Reynolds or Prandtl are large. From the comparison of our results with the results of Massliyah and Epstein [START_REF] Masliyah | Numerical solution of heat and mass transfer from spheroids in steady axi-symmetric flow[END_REF] or those of Juncu [28] we get a perfect match. For a Reynolds number of 100, it is clearly detected, see fig. 8, the occurrence of a minimum of the local Nusselt number for which the polar ɸ angle is 2.5 radians. This angle corresponds to the point of flow separation. We notice, also, that starting from a polar angle of 1.57 radians, corresponding to 90 degrees, the Nusselt number falls and reaches an almost constant value regardless of the Reynolds number. In order to check the behavior of the boundary layer in the vicinity of the particle, we have plotted the variation of the thickness of the boundary layer in this area as a function of Reynolds number (fig. 9).

The variation of the thermal boundary layer is proportional

Results for a spheroid and discussion

Unlike a sphere, an oblate spheroid can be characterized by different equivalent lengths. In this study, we considered the equivalent length to be the principal axis of the spheroid. In order to compare the different results in literature, it is practical to observe the relations given in eqn( 9)

𝑑 𝑒 = 2𝑏 𝐸 2 3 ⁄ =2a 𝐸 -1 3 ⁄ 𝑅𝑒 𝑑𝑒 = 𝑅𝑒 2𝑎 𝐸 1 3 ⁄ 𝑁𝑢 𝑑𝑒 = 𝑁𝑢 2𝑎 𝐸 1 3 ⁄ ( 9
)
A comparison of the numerically obtained drag coefficient was possible to the analytical expression of the drag coefficient developed by Happel and Brenner [START_REF] Happel | Low Reynolds Number Hydrodynamics[END_REF]. The results are compiled in Table 7 for a Reynolds number of 0.1 and for different aspect ratios. This comparison is made for an angle of attack of 0° and 90°. Note that the drag coefficient increases with increasing aspect ratio. There is a maximum of 5% relative difference between numerical and theoretical results. This discrepancy is due to negligence in formulating Happel and Brenner [START_REF] Happel | Low Reynolds Number Hydrodynamics[END_REF] inertial terms.

To validate the numerical results obtained by solving the energy equation, we proceed to compare the change in the local Nusselt number with the results out of the literature. It is the study by Massliyah and Epstein [START_REF] Masliyah | Numerical solution of heat and mass transfer from spheroids in steady axi-symmetric flow[END_REF], which appears to be the first of its kind to have attached importance to the understanding of the mechanism that rules the local heat transfer on the surface of ellipsoidal particles. The authors were able to characterize, through a numerical study, the heat and mass transfer around a spheroidal particle having an aspect ratio of 0.2 for a range of intermediate Reynolds up to 100 and a Prandtl number of 0.7. As for the average heat transfer, they proposed correlations for the average Nusselt number that cannot be used easily due to the complexity of the equivalent length they chose. Indeed, they adopted as equivalent length, the perimeter of the sphere of the same contour as the particle. Following this article, another article by Comer and Kleinstreuer [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF] treated the same subject by considering solid ellipsoids and oblate droplets with an aspect ratio ranging between 0.2 and 1 for a range of intermediate Reynolds numbers ranking between 40 and 120 while the considered Prandtl numbers are 0.7 and 7. Finally, a qualitative study by Juncu [28], described the local heat transfer in axisymmetric steady flow before studying the transitional regime. Two cases were considered. When the heat capacity of the particle is large enough, the temperature is assumed to be constant as it is the case in this study. Otherwise, a spatial variation of the temperature is taken into account. The latter study is conducted on particles of oblate and prolate shape with an aspect ratio ranging between 0.1 and 0.9 in a range of Reynolds number of 10 and 100. The Prandt number is kept equal to 1 and 10. The comparison shows a match between our simulation results and those of Juncu [28], Comer and Kleinstreuer [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF] and Massliyah and Epstein [START_REF] Masliyah | Numerical solution of heat and mass transfer from spheroids in steady axi-symmetric flow[END_REF]. Generally, for a fixed Reynolds and a fixed orientation, the local Nusselt number increases with Prandtl. In addition, the variation of the local Nusselt is not monotonic [START_REF] Srinivas | Numerical analysis of Heat Transfer from a Spheroidal Shaped Body to a Power Law Fluid at Finite Reynolds Number[END_REF]. We note that for an aspect ratio of 0.8, the variation of the local Nusselt number is similar to the case of the sphere to the extent that both admit a maximum at the front stagnation point corresponding to a zero φ. Then, as the aspect ratio decreases, the maximum shifts. This phenomenon is analogous to the reduction of viscous friction when the aspect ratio decreases [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF]. The local Nusselt number exhibited a large value at the front stagnation point and then decreased till the point of separation and then again increased towards the rear stagnation point.

On the other hand, similar to the case of the sphere, the minimum local Nusselt number is observed at the point of separation for the different aspect ratios considered. This observation is clearly visible at higher Peclet number (Fig. 10). By comparing the shape of the plots, with an aspect ratio of 0.2, Renolds of 2 and 100, and Prandtl of 0.7 and 10, note that the separation becomes very visible at large Prandtl numbers. The point of separation away from the stopping point as the aspect ratio increases. And downstream of the point of separation, the local Nusselt number is increasing which is not the case when no separation occurs. In this area, the local Nusselt number is almost independent of the aspect ratio. The fore-aft symmetry is present in the oblate case at very small Reynolds numbers only. This asymmetry leads to wake formation and augments the convective currents resulting in enhanced heat transfer coefficients for intermediate Reynolds numbers.

On another hand, the comparison of our numerical results of the average Nusselt number in a creeping flow shows a good agreement with the results obtained by Brenner [START_REF] Brenner | Forced convection heat and mass transfer at small Péclet numbers from a particle of arbitrary shape[END_REF] in his first order development of the average Nusselt number especially for small Peclet numbers. For this purpose, we had to convert the results obtained from the latter expression to be expressed in terms of equivalent diameter (table 8). With an average error of 1.48%, this relationship takes the geometry shape into account through the aspect ratio but has no consideration of the orientation of the particle. To estimate the precision of the numerical results we obtained for different attack angle, we compared those results at 0° and 90° to the second order development of Brenner [START_REF] Brenner | Forced convection heat and mass transfer at small Péclet numbers from a particle of arbitrary shape[END_REF] valid for an aspect ratio of 0.2 (table 9). The results obtained show that the effect of the orientation increase as the Peclet number become larger. This relation overestimate the average Nusselt number for higher Prandtl numbers and underestimate it at small Prandtl numbers. For aspect ratios different than 0.2 (table 10), the relation given by Massliyah and Epstein [START_REF] Masliyah | Numerical solution of heat and mass transfer from spheroids in steady axi-symmetric flow[END_REF] yields betters results. The accuracy of the average Nusselt number increases when the aspect ratio rises.

For average Reynolds numbers and particle with a null orientation angle, a series of comparisons is presented on fig. 11. The correlation by clift et al., Srinivas and Ramesh [START_REF] Srinivas | Numerical analysis of Heat Transfer from a Spheroidal Shaped Body to a Power Law Fluid at Finite Reynolds Number[END_REF] and Comer and Kleinstreuer [START_REF] Comer | Computational analysis of convection heat transfer to non-spherical particles[END_REF] give reliable results when Prandtl number is equal to 0.7 and 1. Those correlations are suitable for any aspect ratio. However, for a greater Prandtl number, the first two correlations yield poor results while the third authors relations, defined only for specific prandtl numbers of 0.7 and 7, are suitable. In that last case, the results are showing a maximum error of 8% on the domain of Reynolds numbers ranging between 10 and 100. The observations from the comparison with clift et al. [START_REF] Clift | Drops and Particles[END_REF] and Srinivas and Ramesh [START_REF] Srinivas | Numerical analysis of Heat Transfer from a Spheroidal Shaped Body to a Power Law Fluid at Finite Reynolds Number[END_REF], are in agreement with the validity domains of the correlations. In fact, they are valid for Pe2a numbers inferior to 100, Where Pe2a is the Peclet number with equivalent length defined as the great axe of the spheroid. This limit is already reached for a Reynolds number of 10 and a Prandtl number equal to 7 for a small aspect ratio of 0.2. However for a bigger aspect ratio, this limit is not reached yet. The lack of more precise correlations with a larger domain of validity is shown. As for an orientation of 90°(voir fig. 12), comparison was only possible with Kishore and Gu [START_REF] Kishore | Momentum and heat transfer phenomena of spheroid particles at moderate Reynolds and[END_REF] and Sreenivasulu et al. [27]. The first authors, developed an expression which underestimates the average Nusselt number. In fact, even if their correlation was valid in a wide range of Reynolds and Prandtl, it seems less precise than the expression developed by Sreenivasulu et al. especially at higher Reynolds numbers where the maximum error reaches 25% for an aspect ratio of 0.5. The maximum error, according to the researchers can even reach 28%. The relative error diminishes as the aspect ratio gets larger and reaches an average value of 10% for an aspect ratio of 0.8. On the other hand, the mathematical equation developed by Sreenivasulu et al. is only valid for a Prandtl number higher than 5. The comparison shows a good fit between the simulation results and the correlation with a maximal error of 10% in the range of Reynolds numbers between 10 and 100. The precision grows with the Reynolds number and the aspect ratios growth. However, unfortunately, no correlation exists to evaluate the heat transfer around a spheroid with an aspect ratio of 0.2.

The variation of the average Nusselt number is proven to increase with the Reynolds number and the Prandtl number for any aspect ratio considered (fig. 13 and fig. 14). In order to understand the effect of the aspect ratio on the variation of heat transfer, we fixed Reynolds and Prandtl and we plotted the average Nusselt number depending on the aspect ratio (fig. 15). The variation of the Nusselt number depending on the aspect ratio follows a growing non-linear evolution for an orientation of 0° or 90°. A non-monotonic variation is observed in particular for an orientation of 0°. However, if the Nusselt number is recalculated using the major axis of the particle, the variation becomes monotonous (fig. 16). And the more important is the aspect ratio, the more important is the heat transfer if we consider the equivalent length to be the major axis of the oblate spheroid. In case the equivalent length is the minor axis, we get the opposite variation. This aspect shows the importance of the equivalent length on the variation of the Nusselt number.

In order to investigate the effect of the variation of the intermediate angles of attack, we show in fig. 17 the variation of the Average Nusselt number with an equivalent length equal to the major axis, Reynolds of 100 and Prandtl of 7, expressed in terms of the aspect ratio. The figure shows a monotonous variation. It is maximal at 0° and decreases as the angle of attack increases.

Conclusion

A numerical study on heat transfer characteristics has been carried over spheroidal shaped body immersed in a Newtonian incompressible fluid. The ranges of parameters studied consist of a wide range of Reynolds and Prandtl numbers. It was observed that average heat transfer variation over a spheroid depends on the equivalent length we choose. The flow separation that takes place in the oblate spheroid case at Reynolds numbers as low as 30 at 0° orientation is responsible for them having a larger Nusselt number when Reynolds is higher. This flow separation coupled with large Prandtl numbers can yield very high heat transfer coefficients for the oblate spheroids. The variation of heat transfer in terms of orientation of the particle in the fluid is proved to increase when the attack angle is higher. In further works, we will try to develop a simple correlation that one can use to predict the Nusselt number over spheroidal shaped bodies in a wide range of Nusselt and Prandtl numbers. This correlation should also be valid for the case of the sphere.

Stokes

  Those equations are converted into dimensionless form using the equivalent diameter as a reference length, U0 as a reference velocity and as a temperature scale 𝑇 * =

Fig. 2 :

 2 Fig. 2 : Domain of computation and boundary conditions

Fig. 5 :

 5 Fig. 5 : Thermal boundary layer for a Reynolds of 1 and different Prandtl number

Fig. 6 :

 6 Fig. 6 : Local nusselt number distribution for Reynolds of 0.1 and different Prandtl number.

  is the case for a flat plate.

Fig. 7 : 7 Fig. 8 :

 778 Fig. 8 : Variation of local Nusselt number for different Reynolds number and Prandtl number in the case of the sphere

Fig. 10 :

 10 Fig. 10 : Comparaison of the variation of local Nusselt number on the surface of the oblate particle at zero attaque angle for different Reynolds Prandtl and E

Fig. 12 :Fig. 11 :

 1211 Fig. 12 : Comparison of average Nusselt number for an orientation of 90° at high and intermediate Reynolds numbers.

Fig. 13 :Fig. 14 :Fig. 15 :Fig. 16 :Fig. 17 :

 1314151617 Fig. 13 : Variation of the average Nusselt number in terms of aspect ratio for an angle of attack of 90° at fixed Reynolds numbers

Table 1 :

 1 Parameters of simulation

	Aspec	Reynolds	Prandtl	Angle of	Equiv.
	t ratio	number	number	attack	diameter
	E	Re	Pr	α	de
	1	0.1, 1, 10,	0.7, 1, 7	0°	1
		60,100
	0.2,	0.1, 1, 10, 20,	0.7, 1, 7	0° et	1.1696,
	0.5,	40, 60, 80,		90°	1.5874,
	0.8	100			1.8566
	0.2,	1, 60, 100	0.7, 1, 7 15°,30	1.1696,
	0.5,			°,45°,6	1.5874,
	0.8			0°,75°	1.8566

Table 2 :

 2 Domain validation

	Re=0.1
	Diameter of the domain by	Drag coefficient
	particle diameter	D
				C
		d	p	D
	40			247.40
	60			245.43
	80			245.09

Table 3 :

 3 Grid validation

	Re=100, Pr=7 and α=90
				Nu
	Grid A	739749	1.126	12.483
	Grid B	993488	1.122	12.529
	Grid C	3039817	1.119	12.556

Table 4 :

 4 Comparison of drag coefficient in creeping flow

	Re=0.1
		Actual	Stokes [4]	Schiller and Naumann [7]
	CD	247.60	240	247.40

Table 5 :

 5 Comparison of average Nusselt number in creeping flow for Reynolds of 0.1 Another way of validating the results is done by making sure that we really capture the thermal and dynamic boundary layer.

		Nu
	Pr	Numerical	Fourrier	Clift et al.	Friedlander
			[12]	[1]	[16]
	0.7	2.030	2	2.023	2.035
	1	2.043	2	2.032	2.051
	7	2.231	2	2.193	2.461

Table 6 :

 6 Average and maximum relative error between our numerical results of the average Nusselt number and some correlations out of the literature for the case of the sphere

	correlation	Validity domain	Average relatif error (%)	Maximum relatif error (%)
	Feng and Michailides [23]	1<Rep<500 et 1<Pe<1000	1,3	7.03
	Clift et al. [1]	0<Rep<100	1,65	4.39
	Whitaker [21]	1<Rep<100	4,72	9.13
	Li and Mason [22]	0<Rep<100	6,27	11.35

Table 7 :

 7 Comparison of the drag coefficient in the case of oblate spheroid for Reynolds of 0.1 with different aspect ratio and orientation

	CD

Fig. 9 : thermal boundary layer variation with Reynolds number for different Prandtl number

Table 8 :

 8 Comparison of the numerical results of average Nusselt number for a spheroid of aspect ratio of 0.2, 0.5 and 0.8 and a Prandtl number of 0.7,1and 7 at creeping flow with a Reynolds number equal to 0.1

	E
	0.2	0.5	0.8

Table 9 :

 9 Comparison of the numerical results of average Nusselt number for a spheroid of aspect ratio of 0.2 at different orientations and Prandtl numbers at creeping flow with a Reynolds number equal to 0.1

	E = 0.2

Table 10 :

 10 Comparison of the numerical results of average Nusselt number for a spheroid of different aspect ratios and Prandtl numbers at creeping flow with a Reynolds number equal to 0.1

				E
		0.2		0.5		0.8
	Pr	Actual	Massliyah and	Actual	Massliyah and	Actual	Massliyah and
			Epstein [18]		Epstein [18]		Epstein [18]
	0.7	1.559	1.547	1.932	1.923	2.017	2.011
	1	1.571	1.554	1.945	1.932	2.032	2.02
	7	1.731	1.673	2.129	2.082	2.207	2.171
	Average error	1.73%	1.11%		0.84%

May 22nd -27th 2016, Firenze, Italy


Christopher Hernandez
email: hernandezhernandezchristopher@gmail.com

Laura Cadenillas
email: laura.cadenillas.s@gmail.com

Céline Mathieu
email: celine_mathieu@hotmail.com

Jean-Denis Bailly
email: jean-denis.bailly@envt.fr

Vanessa Durrieu
email: vanessa.durrieu@ensiacet.fr

Christopher Hernande

Preservation of Mimosa tenuiflora Antiaflatoxigenic Activity Using Microencapsulation by Spray-Drying

Keywords: encapsulation, spray-drying, antiaflatoxin activity, antioxidant activity, polysaccharides

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Aflatoxin B1 (AFB1) is a worldwide public health issue due to its carcinogenic, immunotoxic and mutagenic properties in humans and animals [START_REF] Iarc | Fungi Producing Significant Mycotoxins[END_REF][START_REF] Wu | Public Health Impacts of Foodborne Mycotoxins[END_REF]. This toxic fungal metabolite is produced by several species of Aspergillus, mostly grouped in the Flavi section [START_REF] Frisvad | Taxonomy of Aspergillus Section Flavi and Their Production of Aflatoxins, Ochratoxins and Other Mycotoxins[END_REF]. Aflatoxins are thermophilic species and AFB1 is a major contaminant in regions with hot climate. It is estimated that almost 4.5 billion people are exposed regularly to this contaminant through their diet, leading to an important number of DALYs (death and disability adjusted life years) [START_REF] Gibb | World Health Organization Estimates of the Global and Regional Disease Burden of Four Foodborne Chemical Toxins, 2010: A Data Synthesis[END_REF]. Moreover, global warming is increasing the geographic repartition of AFB1 that was recorded in regions usually considered as free, such as Europe [START_REF] Bailly | Occurrence and Identification of Aspergillus Section Flavi in the Context of the Emergence of Aflatoxins in French Maize[END_REF]. Aflatoxigenic species can grow and produce toxins either in the field or later during storage, and many crops can be contaminated [START_REF] Waliyar | Reducing Aflatoxins in Groundnuts through Integrated Management and Biocontrol[END_REF][START_REF] Sipos | Physical and Chemical Methods for Reduction in Aflatoxin Content of Feed and Food[END_REF]. Due to the toxicity and especially to the carcinogenic property of AFB1, many countries have set up regulation in different foods to limit consumers' exposure [START_REF]Worldwide Regulations for Mycotoxins in Food and Feed in 2003[END_REF].

Many works are devoted to identifying strategies to limit AFB1 contamination, detoxifying the toxin when present or limiting its deleterious effects after ingestion [START_REF] Sipos | Physical and Chemical Methods for Reduction in Aflatoxin Content of Feed and Food[END_REF].

Due to the great stability of the molecule, the best way to protect consumers from its toxicity seems to be avoiding its presence. Among developing strategies aiming to reduce AFB1 contamination of crops, the use of atoxigenic strains to compete with toxigenic ones was deeply studied [START_REF] Khan | Biocontrol of Aflatoxins Using Non-Aflatoxigenic Aspergillus flavus: A Literature Review[END_REF]. However, this strategy, that leads to interesting results in the short term, could be questionable in the long term due to the risk of recombination between atoxigenic and toxigenic strains [START_REF] Moore | Sex and recombination in aflatoxigenic Aspergilli: Global implications[END_REF]. It also raises the question of its impact on biodiversity.

The use of plant extracts to limit aflatoxigenic species development or AFB1 production is another strategy, and has been increasingly studied over the past 5 years. Several plant extracts, essential oils or isolated components were found able to block aflatoxin B1 production [START_REF] Loi | Plant Bioactive Compounds in Pre-and Postharvest Management for Aflatoxins Reduction[END_REF][START_REF] Peles | Biological control and mitigation of Aflatoxin contamination in commodities[END_REF], among them Mimosa tenuiflora aqueous extract (MAE). Contrary to what is observed using essential oils that inhibit AFB1 synthesis together with reducing fungal growth [START_REF] Gomez | Selected plant essential oils and their main active components, a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food[END_REF], MAE was shown to inhibit AFB1 in A. flavus with no impact on fungal development [START_REF] Hernandez | Mimosa Tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus[END_REF]. This result is interesting because if the extract is able to block AFB1 production without modifying fungal growth, its impact on biodiversity shall be limited. It also reduces the risk of resistance development in target species, since it does not create a selective pressure [START_REF] Bastos | Fungicide effects on human fungal pathogens: Cross resistance to medical drugs and beyond[END_REF]. The effect of MAE was linked to the presence of condensed tannins, in agreement with results observed on mycotoxin production using some wood/forest and vine by-products [START_REF] Montibus | Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins[END_REF].

In many cases, a positive correlation could be demonstrated between antioxidative ability of plant extracts/compounds and their impact on AFB1 synthesis [START_REF] Loi | Plant Bioactive Compounds in Pre-and Postharvest Management for Aflatoxins Reduction[END_REF][START_REF] Hernandez | Mimosa Tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus[END_REF][START_REF] Holmes | Diverse Inhibitors of Aflatoxin Biosynthesis[END_REF][START_REF] Caceres | Piperine Inhibits Aflatoxin B1 Production in Aspergillus Flavus by Modulating Fungal Oxidative Stress Response[END_REF]. This observation raises the question of the preservation of their efficacy with time. Indeed, antioxidative potential can change with time and be reduced due to environmental conditions during storage. Different important factors have been identified, among which are light, temperature, pH, and oxygen [START_REF] Jimenez-Zamora | Antioxidant capacity, total phenols and color profile during the storage of selected plants used for infusion[END_REF][START_REF] Zilic | Effects of baking conditions and dought formulations on phenolic compounds stability, antioxidant capacity and color of cookies made from anthocyanin-rich corn flour[END_REF].

Therefore, the aim of this study was to evaluate the impact of microencapsulation using different wall materials on the antioxidative and antiaflatoxigenic ability of MAE.

Indeed, microencapsulation was recently proposed as a final processing step that can be used to stabilize and protect antioxidant compounds during storage and application [START_REF] Zhang | Microencapsulation of Anthocyanins Extracted from Grape Skin by Emulsification/Internal Gelation Followed by Spray/Freeze-Drying Techniques: Characterization, Stability and Bioaccessibility[END_REF][START_REF] Tomsone | Microencapsulation of Horseradish (Armoracia Rusticana L.) Juice Using Spray-Drying[END_REF]. Through encapsulation, the active ingredients are trapped within the wall materials, limiting their interaction with environmental factors [START_REF] Zhang | Microencapsulation of Anthocyanins Extracted from Grape Skin by Emulsification/Internal Gelation Followed by Spray/Freeze-Drying Techniques: Characterization, Stability and Bioaccessibility[END_REF][START_REF] Dumitrascu | Microencapsulation of Bioactive Compounds from Cornelian Cherry Fruits Using Different Biopolymers with Soy Proteins[END_REF].

This process can also allow for the development of a dry formulation of an active extract. Regarding the limitation of mycotoxin contamination during storage, such dry formulation would be more compatible with its use on dried grains, the active compounds only being released in case of moistening of the medium and subsequent increased risk of mycotoxin production.

Encapsulation depends on several factors, mainly the type of core and wall materials and the technique used. The most widely used encapsulation techniques are spray-drying (SD) and freeze-drying (FD). Different types of biopolymers can be used as wall materials, such as maltodextrin (MD), starch, Arabic gum, and proteins [START_REF] Pereira Souza | Maltodextrin, Pectin and Soy Protein Isolate as Carrier Agents in the Encapsulation of Anthocyanins-Rich Extract from Jaboticaba pomace[END_REF].

Within this context, we encapsulated MAE using the spray-drying method and three different wall materials: maltodextrin, starch, and soy protein. We evaluated the impact of encapsulation process on the antioxidant, antifungal and antiaflatoxigenic effects of MAE. To evaluate the ability of such methods to maintain these activities with time, they were again evaluated after one year of storage at room temperature.

Results and Discussion

Biological Activities of the MAE and Evolution after One-Year Storage (sMAE)

The characterization of the aqueous extract of Mimosa tenuiflora bark at time 0 and after one year of storage at -20 • C (sMAE) is shown in Table 1. After one-year storage, no significant changes in the total amount of phenolic compounds nor the antioxidant activity were observed in sMAE. This may be due to the low temperature of storage (-20 • C), which helps to preserve the stability of the phenolic compounds. However, regarding AFB1 inhibition, the ability of sMAE to inhibit AFB1 production was strongly reduced. Indeed, maximum AFB1 inhibition went from 65.7% at time 0 to 43.8% after one year. This result highlights the need to improve the storage procedure to maintain biological activity of MAE.

MAE Spray-Drying Encapsulation

The process yields obtained during spray-drying step both for microparticles with and without MAE, are shown in Table 2. Soy protein isolate showed the lowest spray-drying yield at 57%, followed by starch (58%) and maltodextrin (59%). The spray-drying yields obtained for the microparticles containing the MAE followed the same trend. As for microparticles without MAE, the highest yield was obtained for maltodextrin-based microparticles (63%), followed by starchbased ones (59%) and soy protein-based ones (54%). Spray-drying yield is not frequently reported for natural compounds. It appears that our results are slightly higher than the few previously reported, such as the 51% described for the encapsulation of aqueous bitter melon extract by spray-drying with maltodextrin [START_REF] Tan | Optimising the Encapsulation of an Aqueous Bitter Melon Extract by Spray-Drying[END_REF], or the 49% in the case of microparticles prepared with OSA starch to encapsulate Jussara pulp [START_REF] Lacerda | Inulin and Maltodextrin as Encapsulating Agents Affect the Quality and Stability of Jussara Pulp Microparticles[END_REF].

These yields can be considered promising regarding the use of this process at an industrial scale for the protection of bioactive compounds, especially considering the low amount of dry matter used during the atomization step.

Particles Characterization 2.3.1. Particles Morphology and Size

The macroscopic aspect of the produced microparticles is shown in Figure 1. The microparticles produced using maltodextrin were the most homogeneous and appeared lighter in color than the others (Figure 1A). When MAE was incorporated, a brown color was observed for all wall materials (Figure 1D-F). As noted for microparticles made only with support material, those made with maltodextrin appeared mildly lighter and thinner than the others (Figure 1D).

A morphological analysis was carried out to evaluate the characteristics of the microparticles such as shape, surface, as well as their size distribution (Figure 2). Scanning electron microscopy showed that the microparticles are globally spherical, heterogeneous in size and not agglomerated, which is probably related to repulsion by negative charges.

Figure 2 shows that the microparticles had a wrinkled surface, probably because they collapsed in their center. This observation can be explained by the evaporation of water contained in droplets inside the drying chamber during the spraying step [START_REF] Simon-Brown | Microencapsulation of Ginger (Zingiber officinale) Extract by Spray Drying Technology[END_REF]. This phenomenon was previously observed in plant protein-based microparticles with low dry matter content [START_REF] Nesterenko | A New Way of Valorizing Biomaterials: The Use of Sunflower Protein for α-Tocopherol Microencapsulation[END_REF]. This could also be related to SEM analysis, which is performed under vacuum which can deform the microparticles [START_REF] Ribeiro | Microencapsulation of Polyphenols-The Specific Case of the Microencapsulation of Sambucus Nigra L. Extracts-A Review[END_REF]. The sizes of microparticles made using polysaccharides as wall material (maltodextrin and starch) were about 3 µm in size. Those based on the use of soy protein were larger and had a less homogeneous size distribution, ranging between 10 and 15 µm and reaching up to 30 µm in some cases. There could be a link between the intrinsic size of the matrix constituent macromolecules and the size of the resulting microparticles. Furthermore, soy protein was more difficult to dissolve than the others; the large particle visible on Figure 2C could be the result of this incomplete dissolution.

Total Polyphenol Content and Antioxidant Activity of Wall Material Microparticles

The total polyphenol content and antioxidant activity of the particles made with wall materials only are presented on Table 3. None of the microparticles with support material presented antioxidant activity. Total polyphenol content in microparticles made with the two polysaccharides (maltodextrin and starch) was not significant. By contrast, the soy protein microparticles showed a polyphenol content of 17 mg GAE/g DM. This value is attributed to the fact that the Folin reagent, used for polyphenol determination, can also react with proteins, as used in the Lowry method for protein determination [START_REF] Shen | Quantification and Analysis of Proteins[END_REF]. However, this value was low compared to values measured in MAE. Moreover, if due to protein interactions, this value shall remain constant and not interfere with the evaluation of the evolution of polyphenol content in microparticles containing MAE with storage time.

Total Polyphenol Content and Antioxidant Capacity of Microparticles with MAE and Evolution after 1 Year of Storage

Table 4 gathers the results of the total polyphenols and antioxidant capacity of the microparticles containing MAE on time 0 and after one year of storage at room temperature. The determination of the polyphenols encapsulation efficiency (PEE) allowed us to demonstrate the capacity of the tested wall materials to preserve the polyphenols during the atomization stage. For all the microparticles, the experimental results of the polyphenol determination were lower than the theoretical ones. This could be explained by the matter loss on the apparatus walls during the encapsulation process (both some wall material and compounds belonging to the extract).

Polysaccharide-based microparticles had the highest polyphenols encapsulation efficiency, with 86% and 65% for starch and maltodextrin, respectively. Soy protein-based ones had the lowest efficiency, with 58%. This difference may be due to the polysaccharides' hydrophilic character, making their affinity for an aqueous extract higher than that of soy protein, which is more hydrophobic. The difference between the two polysaccharides may be related to the longer chain molecular structure of starch that can improve the phenolic compounds protection. The microparticles' storage for one year at room temperature did not significantly modify the polyphenol content, whatever the wall material considered.

Only few data are available concerning the encapsulation of natural extracts. Nevertheless, we can compare our results with those obtained during the microencapsulation of phenolic compounds of pomegranate juice by spray-drying, where the microparticles generated with maltodextrin presented a lower polyphenol encapsulation efficiency (54%). By contrast, the microparticles generated with soy protein presented a higher efficiency (76%) [START_REF] Robert | Encapsulation of Polyphenols and Anthocyanins from Pomegranate (Punica Granatum) by Spray Drying[END_REF]. Our results follow the same trend as those reported for the encapsulation of polyphenols of horseradish juice by spray-drying with maltodextrin (75%), starch (77%) and soy protein (56%) [START_REF] Tomsone | Microencapsulation of Horseradish (Armoracia Rusticana L.) Juice Using Spray-Drying[END_REF]. Our results are also comparable with the results described for anthocyanins' encapsulation by spray-drying with sodium alginate as wall material (75%) [START_REF] Zhang | Microencapsulation of Anthocyanins Extracted from Grape Skin by Emulsification/Internal Gelation Followed by Spray/Freeze-Drying Techniques: Characterization, Stability and Bioaccessibility[END_REF] and of carotenoids by spray-drying with a mixture of maltodextrin and Arabic gum, which presented an efficiency of 77% [START_REF] Ferreira | Green Extraction by Ultrasound, Microencapsulation by Spray Drying and Antioxidant Activity of the Tucuma Coproduct (Astrocaryum Vulgare Mart.) Almonds[END_REF].

The antioxidant capacity of MAE was not modified by encapsulation with the two polysaccharide-based microparticles. Indeed, considering the 50/50 ratio (w/w) between the extract and the wall material used for encapsulation, which led to a division of the DM of MAE by two-fold in the microparticles, it seems that the initial antioxidant activity of the aqueous extract was preserved. Moreover, it was not significantly affected by one-year storage at room temperature (Table 4). By contrast, the antioxidant activity of MAE in soy protein-based microparticles was twice lower than that for polysaccharides. This may be due to the fact that antioxidant compounds can interact with proteins, reducing their activity. This observation is even more important after one year of storage, since antioxidant activity decreased again about two-fold, increasing the difference to polysaccharide-based microparticles.

Antifungal and Antiaflatoxin Activities 2.4.1. Antifungal and Antiaflatoxin Activities of the Wall Material Microparticles

Figure 3 shows the effect of microparticles prepared with wall materials only (no MAE) on the growth and AFB1 production by A. flavus NRLL 62477 strain. The addition of increasing concentrations of wall material microparticles mildly modified the growth of A. flavus, likely related to the modification of available nutrients in the medium. Maltodextrin led to a slight reduction in growth (less than 4% at the highest tested concentration) whereas the two other wall materials mildly increased fungal growth. However, this had no significant impact on AFB1 production, with the exception of the highest concentration of soy protein microparticles (0.2 mg DM/mL), that slightly increased AFB1 production after 8 days of culture. This can be directly related to the increased fungal development observed at this concentration.

Antifungal and Antiaflatoxin Activities of the Microparticles with MAE and Evolution after One Year pf Storage

The impact of MAE-containing microparticles on both fungal growth and AFB1 production at T0 and after one-year storage are presented on Figure 4. The impact of MAE-containing microparticles on fungal growth at T0 was very limited (Figure 4A). Indeed, both starch and soy protein had no effect whatever the concentration tested. Only maltodextrin microparticles mildly reduced fungal growth (less than 5%) at all tested concentrations, as observed previously for particles without MAE. After oneyear storage, quite comparable results were observed with no effect on fungal growth for both starch and maltodextrin at all tested concentrations. We were not able to test MAEcontaining soy-protein microparticles after one year due to a lack of particles. Therefore, encapsulation of MAE in the different microparticles did not modify its limited impact on fungal growth.

Figure 4C shows the impact of MAE-containing microparticles on AFB1 production in A. flavus, compared to the effect of MAE at T0. Microparticles prepared with both polysaccharides had a similar inhibitory effect on AFB1 to MAE. Soy protein-based microcapsules were mildly less efficient in AFB1 inhibition. This could be the result of the balance between the stimulation of AFB1 production observed with soy protein alone (Figure 3B) and the effect of MAE active compounds on AFB1 synthesis.

After one year of storage, MAE-containing microparticles prepared with starch and maltodextrin were still able to inhibit AFB1 synthesis in a dose-dependent manner, with no significant reduction in their efficacy compared to what was observed at T0. By contrast, MAE extract stored for the same time at -20 • C lost part of its activity, since the maximal AFB1 inhibition was about 40% whereas it was higher than 65% at time 0.

In this study, we demonstrated that polysaccharide microparticles were able to preserve anti-AFB1 activity of MAE more efficiently than after storage at -20 • C. Since microparticles can be stored at room temperature with no special constraints, this may represent a good and energy-saving method of long-term storage of active components to be used on crops. The dry formulation is also of great interest considering grain protection during storage, meaning after a drying step. Indeed, in that case, active molecules from MAE would be released only in cases of moistening, which corresponds to an increase in the risk of fungal development and of subsequent toxin production.

Materials and Methods

Chemicals, Reagents and Encapsulating Agents

All solvents (HPLC grade) (acetonitrile; chloroform and methanol) and chemicals (Tween 80; Folin-Ciocalteu reagent; DPPH reagent; AFB1 standard; starch) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Ethanol 96% and sodium carbonate were purchased from VWR International (Fontenay-sous-bois, France). Ultrapure water was prepared using Veolia Purelab Classic (Veolia, Toulouse, France). Maltodextrin Glucidex IT12 was purchased from Roquette (Lestrem, France) and soy protein SUPRO ® XT219DIP from Solae Belgium NV (Ieper, Belgium).

Plant Material

The supplier of stem bark of M. tenuiflora (Willd.) was Red Mexicana de Plantas Medicinales y Aromaticas (REDMEXPLAM) under the registration number UATX/01/ Tepezcohuite/2019 deposited at Jardin Botanico Universitario UAT. At reception, bark was ground at 1 mm granulometry using a mill and then stored at 4 • C in sealed container.

Preparation of M. tenuiflora Aqueous Extract (MAE)

MAE was prepared as described [START_REF] Hernandez | Mimosa Tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus[END_REF]. M. tenuiflora bark was extracted by maceration for 15 h at room temperature. The resultant extract after centrifugation was filtered through Whatmann grade 1 filter paper (Sigma-Aldrich, Saint-Quentin-Fallavier, France). The sterilization was carried out at 121 • C for 20 min in an autoclave (SMI group UNICOM, Montpellier, France).

Dry Matter Content

Dry matter contents were determined for the extract and encapsulating agents by placing them at 103 • C until constant weight, using a Memmert oven (Schabach, Germany).

MAE Encapsulation by Spray-Drying

Infeed solutions were prepared as follows for all the encapsulating materials: the wall material was added to MAE under mechanical stirring (500 rpm) and the solution was heated at 70 • C for 30 min (still under stirring) to facilitate the solubilization of the wall material powder. The infeed solutions contained 0.65% (w/w) total solids, for a 50:50 core-to-wall ratio.

Freshly prepared solutions were then spray-dried using a Mini Spray-Dryer B-290 (Büchi, Flawil, Switzerland), in the open mode under the following stable conditions: inlet air temperature of 140 ± 4 • C, drying airflow rate of 470 L/h, liquid feed flow rate of 450 mL/h and 100% aspiration. Infeed solutions were maintained under magnetic stirring during the entire spray-drying step. Microparticles collected from the container were sealed in a hermetic opaque packaging and stored for analysis at room temperature. The spray-drying yield was calculated as follows:

SP yield = [mass of microparticles recovered/Initial solid dry mass] × 100,

(1)

Polyphenol Encapsulation Efficiency (PEE)

The encapsulation efficiency of the polyphenols was calculated as the experimental content of polyphenols contained in the microparticles in relation to the theoretical content of polyphenols. %PEE = [Experimental content of polyphenols in microparticles/Theoretical content of polyphenols in microparticle] × 100 [START_REF] Wu | Public Health Impacts of Foodborne Mycotoxins[END_REF] The theoretical polyphenol content in the microparticles was calculated according to the polyphenol content of the extract and its proportional dilution in the wall material.

Scanning Electron Microscopy (SEM) Analysis

SEM observations of microparticles were performed using a Quanta 450 FEG scanning electron microscope (FEI, Hillsboro, OR, USA) under low vacuum mode at 100 Pa. The microparticles were deposited on conductive double-faced adhesive tape and sputtercoated with silver. They were observed at different magnifications (1000× and 8000×).

Solubilization of Microparticles for Analyses

The three wall materials are soluble in water, but not very soluble in ethanol. Therefore, the microparticles were first diluted in water, to carry out the tests on the fungus. For the determination of the antioxidant capacity, they were rediluted in ethanol as required by the technique, due to the solubility of the DPPH reagent in ethanol.

Determination of Total Polyphenol Content

The Folin-Ciocalteu method [START_REF] Singleton | Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents[END_REF] was applied for the determination of total polyphenols content. The absorbance reading was performed at 700 nm after 45 min incubation time at 45 • C using a spectrophotometer BMG-Labtech SpectrostarNano (BMG LABTECH SARL, Champigny-sur-Marne, France). The results were expressed in milligrams of Gallic Acid Equivalent (GAE) per gram of dry matter.

Determination of Antioxidant Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich) free radical was used to determine antioxidant activity of extracts and microparticles [START_REF] Brand-Williams | Use of a Free Radical Method to Evaluate Antioxidant Activity[END_REF]. The reagent solution of DPPH in ethanol 96% was adjusted at a 516 nm absorbance of 1+/-10% in the same analyzing conditions as the extracts on BMG-LabtechSpectrostar-Nanospectrophotometer (BMG LABTECH SARL, Champigny-sur-Marne, France).

Seven different extract concentrations (150 µL) were incubated with 150 µL DPPH solution for 40 min at room temperature in a 96-well quartz microplate. The reaction equation for each reaction solution of five replicates was:

f (Ce)c = 1 -A/A 0 ( 3
)
where Ce is the extract concentration (in mg/L), A the extract absorbance, and A 0, is the control absorbance (DPPH) after 40 min incubation. The antioxidant activity is reported as the inhibitory concentration 50 (IC 50 ), which corresponds to the concentration that inhibits 50% of the DPPH radicals. It is expressed in milligrams of extract per liter (mg/L). The value of IC50 was calculated using linear regression, as follows:

IC 50 = 0.5 -a/b (4)
where IC 50 is the half-maximal inhibitory concentration, a is the origin ordinate, and b is the slope. The strain used for all assays was Aspergillus flavus NRRL 62477 [START_REF] El Mahgubi | Distribution and Toxigenicity of Aspergillus Section Flavi in Spices Marketed in Morocco[END_REF]. Ten µL of a spore suspension containing 1000 spores were inoculated centrally into the culture medium for all experiments [START_REF] Hernandez | Mimosa Tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus[END_REF].

The culture medium was made of 18 mL of malt extract agar (Biokar Diagnostics, Allonne, France) and 2 mL of autoclaved MAE or microparticles extracts, prepared at different concentrations by dilution in water. Control cultures were made by adding 2 mL of distilled water to the initial 18 mL of culture medium. Cultures were incubated for 8 days at 27 • C. Each assay was performed in triplicate. After incubation, the growth was evaluated by the measurement of the colony diameter.

3.11.2. Extraction and Quantification of AFB1 by HPLC AFB1 was extracted from culture media with HPLC-grade chloroform and the supernatants were filtered off. Filtrates (2 mL) were concentrated to dryness at 45 • C using a STUART SBH200D/3 sample concentrator (Stuart equipment, Paris, France), and the residues were redissolved in acetonitrile HPLC grade (2 mL). Finally, the samples were filtered through 0.45 µm PTFE disk filters (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) and placed in HPLC vials. AFB1 concentration was quantified by HPLC on an Ultimate 3000 UPLC equipment coupled to fluorescent detector at 365 nm wavelengths for excitation (and 430 nm for emission) (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). Separation was carried out with an EvoC18 column (3 µm, 150 × 3.2, Phenomenex, Le Pecq, France) at 27 • C, at a 1.2 mL/min flow of a mixture acetonitrile HPLC grade and water (25:75 v:v). Injection volume was 10 µL.

The identity of the AFB1 was confirmed by checking the UV absorption spectrum thanks to an additional DAD detector in the series. AFB1 concentrations were calculated with an external standard calibration curve in the range of 0.16 to 20 mg/L.

Statistical Analysis

All data are presented as the mean of the experimental values obtained in quadruplicate. The error values of the data shown in tables as numerical values or in graphs as error bars correspond to the standard error of the mean (SEM) calculated from the standard deviation of the values.

One-way ANOVA was used to determine the differences in the AFB1 production and fungal growth data between the control and treated groups. A T-test was used to determine the significant difference between the means of two groups.

Differences were considered statistically significant for p-values less than 0.05. GraphPad Prism 9 software was used to perform all statistical analyzes.

Conclusions

This work demonstrates that microencapsulation is a promising process to maintain the antioxidant and antiaflatoxigenic properties of natural extracts during long periods of storage. Among the wall materials tested here, polysaccharides appear to be the best choice since they are neutral on both fungal growth and toxinogenesis. Further studies are now required to test the applicability of such microparticles on grains during storage.

Figure 1 .

 1 Figure 1. Visual aspect of the powders obtained after atomization in Mini Spray-dryer Buchi B-290 of wall materials alone: maltodextrin (A), soy protein (B), starch (C). Powders obtained after atomization of wall materials together with MAE are presented on the second line: maltodextrin + MAE (D), soy protein + MAE (E), and starch + MAE (F).

Figure 2 .

 2 Figure 2. Microparticle analysis by SEM. Observations with a Scanning Electron Microscope of the microparticles obtained after MAE encapsulation in the three wall materials. (A,B) Maltodextrin microparticles with MAE, (C,D) soy protein microparticles with MAE, (E,F) starch microparticles with MAE.

Figure 3 .

 3 Figure 3. Fungal growth (A) and AFB1 production (B) of A. flavus NRRL 62477 strain when exposed to increasing concentrations of the different wall-material microparticles for 8 days at 25 • C: Starch (blue bars), maltodextrin (green bars) and soy protein (yellow bars). Columns with 0 correspond to A. flavus NRRL 62477 cultures with no wall materials and are used as control values. Results are expressed as the percentage of fungal growth or AFB1 production compared to untreated control cultures ± standard error of the mean (n = 4). ns = no significant change; * p-value < 0.1; ** p-value < 0.01; *** p-value < 0.001.

Figure 4 .

 4 Figure 4. Fungal growth at time 0 (A) and after one-year storage (B). AFB1 production at time 0 (C) and after one-year storage (D). A. flavus NRRL 62477 strain was exposed to increasing concentrations of MAE-containing microparticles for 8 days at 27 • C. MAE (A,C) and sMAE (B,D) were used as a positive control at T0 and after one year of storage, respectively. Columns with 0 correspond to A. flavus NRRL 62477 cultures with no particles nor MAE or same, and are used as control values. Results are expressed as the percentage of fungal growth or AFB1 production compared to these untreated control cultures ± standard error of the mean (n = 4). ns = no significant change; * p-value < 0.1; ** p-value < 0.01; *** p-value < 0.001.

3. 11 .

 11 Effect of MAE and Microparticles on Aspergillus Flavus Growth and Aflatoxin B1 Synthesis 3.11.1. Fungal Strain and Culture Conditions

Table 1 .

 1 Characterization of the aqueous extract at time 0 and after one year of storage at -20 • C.

Dry Mass (%) Polyphenol Content (mg GAE/g DM Extract) Antioxidant Activity b IC50 (mg/L) Maximum AFB1 Inhibition c (%) Maximum Fungal Inhibition c (%)

	MAE	11.2	397 ± 22	10	65.7	3
	sMAE a	11.1	376 ± 2 ns	12	43.8 *	1 ns
			ns no statistically significant change; * statistically significant change: p-value < 0.05. a : sMAE: MAE stored one
			year at -20 • C. b : measured using DDPH assay. c measured after incubation of Aspergillus flavus NRRL 62477
			with 0.2 mg DM/mL for 8 days at 27 • C.

Table 2 .

 2 Spray-drying yields for the different wall materials tested.

Composition of the Microparticles Wall Material/Extract Ratio (w/w Dry Matter) Spray-Drying Yield (%)

	Maltodextrin	100/0	59
	Maltodextrin + MAE	50/50	63
	Soy protein	100/0	57
	Soy protein + MAE	50/50	54
	Starch	100/0	58
	Starch + MAE	50/50	59

Table 3 .

 3 Total polyphenol content and antioxidant activity of wall material microparticles.

	Wall Material	Polyphenol Content (mg GAE/g DM)	Antioxidant Activity on DPPH IC50 (mg DM/L)
	Maltodextrin	0.7 ± 0.3	>1000
	Soy protein	17.0 ± 0.4	>1000
	Starch	1.9 ± 0.4	>1000

Table 4 .

 4 Polyphenol content and antioxidant activity of the microparticles with MAE and evolution after one-year storage at room temperature.

	Wall Material Used	Theoretical Dry Mass of the Extract in the Microparticles (g)	Theoretical Polyphenol Content in Microparticles (mg GAE/g Microparticles)	Experimental Microparticles) Polyphenol Content in Microparticles (mg GAE/g	Time 0 PEE a (%)	Antioxidant Activity IC50 b (mg/L)	After One-Year Storage c Experimental Microparticles) Polyphenol Antioxidant Content in Activity IC50 Microparticles (mg GAE/g b (mg/L)
	Maltodextrin	0.65	258	168 ± 7	65	23	188 ± 3	27
	Starch	0.62	246	214 ± 8	86	25	211 ± 3	27
	Soy protein	0.62	246	142 ± 9	58	48	149 ± 3	79

a : PEE = Polyphenols encapsulation efficiency. b : measured using DDPH assay. c : storage at room temperature.

Data Availability Statement:

The data presented in this study are available on request to the corresponding author.

Conflicts of Interest:

The authors declare no conflict of interest.

Sample Availability: Not applicable.