Matthieu Rivière

Ronan Cariou

Sébastien Hutinet

Asmaa Tebbaa

Yann Guitton

Didier Dubreuil

Monique Mathe-Allainmat

Jacques Lebreton

Bruno Le Bizec

Arnaud Tessier

Non-volatile substances extractible from inner coatings of metallic cans from the French market and their occurrence in the canned vegetables

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

Introduction Experimental

Results and Discussion Conclusion www.laberca.org 2 Nantes Université, CNRS, CEISAM, UMR 6230, F-44000 Nantes, France

Ronan CARIOU 1 , Matthieu RIVIÈRE 2 , Sébastien HUTINET 1 , Asmaa TEBBAA 1,2 , Yann GUITTON 1 , Didier DUBREUIL 2 , Monique MATHE ALLAINMAT 2 , Jacques LEBRETON 2 , Bruno LE BIZEC 1 , Arnaud TESSIER 2 , Gaud DERVILLY 1

Non-volatile substances extractible from inner coatings of metallic cans from the French market and their occurrence in the canned vegetables Using a non-targeted LC/ESI(+)-HRMS approach, dozens of small molecules in inner coatings of vegetable metallic cans have been identified. Oligoesters, a diverse family of unwanted compounds arising from the synthesis of polyesters, prevailed in the data set.

This study sheds light on the need to fulfil a proper risk assessment on this NIAS family (exposure and hazard characterisation, including genotoxicity and endocrine disruption).

Detailed information in DOI 10.1016/j.jhazmat.2022.129026.

  Regulatory pressure against bisphenol A for food contact material (FCM) applications led can coating manufacturers to develop polyester-based alternatives. Besides intentionally added substances, the investigation of the presence of non-intentionally added substances (NIAS) has been raising the interest of the scientific community in response to manufacturers and consumers concerns. NIAS, generally of unknown structures, can be impurities from starting materials, degradation or decomposition products, contaminants from production process or oligomers principally formed during the synthesis of the coating. Such oligomers can be formed during resin synthesis or upon degradation over time. Although a wide range of reactions are possible (which poses an analytical challenge), these NIAS may be predicted since polyalcohols and carboxylic polyacids involved in the polycondensation reaction are generally known. In order to increase chemical food safety of packaging, it is now necessary to produce qualitative, quantitative and toxicological data.This work aimed to contribute to the risk assessment of NIAS from polyester-based coatings by developing an innovative analytical strategy dedicated to their characterisation.

Figure 1 :

 1 Figure 1: Study design.

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: Stepwise synthesis of native and D-labelled combinations of NPG and iPA.

Figure 5 :Figure 6 :O

 56 Figure 5: Oligoesters observed (84 monomer combinations; 38 out of the 40 coatings).


Laurence Pessoni
email: laurence.pessoni@univ-pau.fr

David Siniscalco

Anne Boussonnière

Anne-Sophie Castanet

Laurent Billon

Nicolas Delorme
email: nicolas.delorme@univ-lemans.fr

Photo-reversible solid to liquid transition of azobenzene containing polymers: impact of the chemical structure and chain length

Keywords: azobenzene, nitroxide mediated polymerization, azopolymer, isomerization, solid-to-liquid transition

Azobenzene containing polymers (azopolymers) can exhibit a photo-reversible solid to liquid transition which is illustrated by a cyclic softening and hardening of the polymer material upon irradiation. In order to understand the structure-property relationship on this transition, Nitroxide Mediated Polymerization (NMP) was used for the first time to synthesize a series of azopolymers.

Four azopolymers bearing various azobenzene groups were designed with different molecular weights. Photo-isomerization properties and photo-reversible solid-to-liquid transitions were studied as function of the molecular weight and structure of the azopolymers. It was found that molecular weights and chemical structure of the azopolymer have both an impact on the features of the reversible solid-to-liquid transition. In particular, the higher the length of the substituent or the linker, the faster the transition. The results obtained in this study help on the one hand to better understand the mechanism of the photo-reversible solid-to-liquid transition and on the other hand to consider the design of novel azopolymers for many applications.

Introduction

There is an increasing demand in the industry for smart materials and coatings that exhibit advanced properties as reversible responses to environmental changes, [START_REF] Huang | Stimuli-Responsive Surfaces for Tunable and Reversible Control of Wettability[END_REF] including for example: temperature, [START_REF] Ye | Fabrication of a Self-Cleaning Surface via the Thermosensitive Copolymer Brush of P(NIPAAm-PEGMA)[END_REF] CO 2 , [START_REF] Marcasuzaa | CO2-Driven reversible wettability in a reactive hierarchically patterned bio-inspired honeycomb film[END_REF][START_REF] Yin | CO2-Triggered ON/OFF Wettability Switching on Bioinspired Polylactic Acid Porous Films for Controllable Bioadhesion[END_REF] pH [START_REF] Marcasuzaa | When a pH-triggered nanopatterned shape transition drives the wettability of a hierarchically self-organized film: A bio-inspired effect of "sea Anemone[END_REF]. Light-responsive materials in which a quick and reversible response can be achieved are of particular interest. Indeed, light is a non-contact and mild energy source, which can be used specifically with regard to wavelength, intensity, or polarization direction to achieve precise and remote control. [START_REF] Manrique-Juárez | Switchable molecule-based materials for micro-and nanoscale actuating applications: Achievements and prospects[END_REF] It has been known for years that depending of their molecular structure, azobenzene molecules and their derivatives can undergo reversible trans to cis isomerization upon irradiation with UV or visible light. [START_REF] Horspool | CRC handbook of: Organic photochemistry and photobiology[END_REF][START_REF] Dürr | Photochromism: Molecules and systems[END_REF][START_REF] Rau | On the Rotation-Inversion Controversy on Photoisomerization of Azobenzenes. Experimental Proof of Inversion[END_REF] Azopolymers are well known for their efficient and robust photo-chemistry. [START_REF] Gondry | Other Cis-Trans Isomerizations in Organic Molecules and Biomolecules[END_REF] For these reasons, they have found applications in several fields [START_REF] Wang | Azo Polymers[END_REF] such as biomedical application, [START_REF] Ji | Photosensitive hydrogels: from structure, mechanisms, design to bioapplications[END_REF][START_REF] Cheng | Advances in Application of Azobenzene as a Trigger in Biomedicine: Molecular Design and Spontaneous Assembly[END_REF] in photoresponsive molecularly imprinted polymers (PMIPs), [START_REF] Wei | Review of the recent progress in photoresponsive molecularly imprinted polymers containing azobenzene chromophores[END_REF] actuators, [START_REF] Kim | Shape morphing smart 3D actuator materials for micro soft robot[END_REF][START_REF] Yu | Directed bending of a polymer film by light[END_REF] information storage, [START_REF] Ikeda | Optical switching and image storage by means of azobenzene liquid-crystal films[END_REF][START_REF] Colquhoun | Self-repairing polymers: Materials that heal themselves[END_REF] solar energy storage, [START_REF] Wu | Solar-Thermal Energy Conversion and Storage Using Photoresponsive Azobenzene-Containing Polymers[END_REF] or light-controlled microfluidics and other photonic devices. [START_REF] Priimagi | Azopolymer-based micro-and nanopatterning for photonic applications[END_REF][START_REF] Ikeda | Photomechanics of liquid-crystalline elastomers and other polymers[END_REF][START_REF] Palagi | Structured light enables biomimetic swimming and versatile locomotion of photoresponsive soft microrobots[END_REF][START_REF] Xu | Light-Directed Liquid Manipulation in Flexible Bilayer Microtubes[END_REF] Recently, it has been shown for some azopolymers that the reversible trans-cis photo-isomerization of the azobenzene moiety induces a reversible change on the mechanical properties of the material called Reversible Solid-to-Liquid Phase Transition (RSLPT) or photofluidization. [START_REF] Li | Photoinduced healing of mechanically robust polymers[END_REF][START_REF] Xu | Photoswitchable Adhesives Using Azobenzene-Containing Materials[END_REF][START_REF] Xu | Photoinduced Reversible Solid-to-Liquid Transitions for Photoswitchable Materials[END_REF][START_REF] Weis | Photoinduced Liquefaction of Azobenzene-Containing Polymers[END_REF] This transition is illustrated by a repeatable softening and hardening of the polymer material upon irradiation.

Since then, the development of polymers with switchable mechanical properties has become an emerging research field in materials science and technology because of its potential applications such as adhesives, [START_REF] Xu | Photoswitchable Adhesives Using Azobenzene-Containing Materials[END_REF] self-healing materials, [START_REF] Yamamoto | Photochemical liquefaction and softening in molecular materials, polymers, and related compounds[END_REF] photolithography [START_REF] Yang | Azopolymer-Based Nanoimprint Lithography: Recent Developments in Methodology and Applications[END_REF][START_REF] Oscurato Stefano | From nanoscopic to macroscopic photodriven motion in azobenzene-containing materials[END_REF] or anti-bacterial coating. [START_REF] Kehe | Optically Responsive, Smart Anti-Bacterial Coatings via the Photofluidization of Azobenzenes[END_REF] Although, the RSLPT mechanism was rarely investigated, [START_REF] Xu | Photoinduced Reversible Solid-to-Liquid Transitions for Photoswitchable Materials[END_REF] the main hypothesis is that RSLPT is linked to a glass temperature variation during the trans-cis photo-isomerization. For polymers, RSLPT may occur when the processing temperature is between the glass transition temperature (i.e. Tg) of the cis form and that of the trans form. For instance, for an azopolymer with an acrylate backbone showing a RSLPT in ambient conditions, Zhou et al. [START_REF] Zhou | Photoswitching of glass transition temperatures of azobenzene-containing polymers induces reversible solid-to-liquid transitions[END_REF] measured by DSC a decrease from Tg trans = 48°C to Tg cis = -10°C upon photo-isomerization.

Until now, only a few photo-sensitive polymers that undergo a RSLPT have been reported. [START_REF] Yue | Light-induced mechanical response in crosslinked liquidcrystalline polymers with photoswitchable glass transition temperatures[END_REF] Most of these polymers are based on poly(meth)acrylates backbones [START_REF] Yamamoto | Photochemical liquefaction and softening in molecular materials, polymers, and related compounds[END_REF][START_REF] Zhou | Photoswitching of glass transition temperatures of azobenzene-containing polymers induces reversible solid-to-liquid transitions[END_REF][START_REF] Xu | Influence of chromophoric electron-donating groups on photoinduced solid-to-liquid transitions of azopolymers[END_REF][START_REF] Ito | Azobenzene-Based (Meth)acrylates: Controlled Radical Polymerization, Photoresponsive Solid-Liquid Phase Transition Behavior, and Application to Reworkable Adhesives[END_REF][START_REF] Cieciórski | New photoresponsive poly(Meth)acrylates bearing azobenzene moieties obtained via atrp polymerization exhibiting liquid-crystalline behavior[END_REF][START_REF] Weis | Effects of Spacers on Photoinduced Reversible Solid-to-Liquid Transitions of Azobenzene-Containing Polymers[END_REF][START_REF] Akiyama | Reworkable adhesives composed of photoresponsive azobenzene polymer for glass substrates[END_REF] with azobenzene type molecules in the side chain. One of the reasons of the low number of available polymers that undergo RSLPT, is the strong influence of the azobenzene compounds structure on the isomerization properties. For example, Wu and co-workers in 2019 [START_REF] Weis | Effects of Spacers on Photoinduced Reversible Solid-to-Liquid Transitions of Azobenzene-Containing Polymers[END_REF] have shown that the azobenzene/polymer backbone spacer plays a major role in the RSLPT of poly(meth)acrylates azo homopolymers. Indeed, for short chain lengths, the Tg did not show a significant decrease after irradiation and the phase transition temperature was above room temperature for higher lengths. More recently, in 2020, Xu et al. [START_REF] Xu | Influence of chromophoric electron-donating groups on photoinduced solid-to-liquid transitions of azopolymers[END_REF] have demonstrated that the liquefaction rate of azopolymers drastically increased as the size of substituents increased. Despite these studies, there are still several critical issues or lack that need to be addressed in order to further elucidate the correlation between the macromolecular features, i.e. nature of the azo-monomer, length of the polymer…. and the photo-responsiveness of azopolymers.

Properties of photoresponsive azopolymers depend indeed on their molecular weight and dispersity Đ values . [START_REF] Wang | Azo Polymers[END_REF] We have then chosen controlled radical polymerization CRP allowing simultaneously the control of the number-average molar mass, the dispersity and the chain end of polymer. [START_REF] Corrigan | Reversible-deactivation radical polymerization (Controlled/living radical polymerization): From discovery to materials design and applications[END_REF] ATRP, [START_REF] Xu | Influence of chromophoric electron-donating groups on photoinduced solid-to-liquid transitions of azopolymers[END_REF][START_REF] Ito | Azobenzene-Based (Meth)acrylates: Controlled Radical Polymerization, Photoresponsive Solid-Liquid Phase Transition Behavior, and Application to Reworkable Adhesives[END_REF][START_REF] Cieciórski | New photoresponsive poly(Meth)acrylates bearing azobenzene moieties obtained via atrp polymerization exhibiting liquid-crystalline behavior[END_REF][START_REF] Chen | Entangled Azobenzene-Containing Polymers with Photoinduced Reversible Solid-to-Liquid Transitions for Healable and Reprocessable Photoactuators[END_REF][START_REF] Pessoni | Light-triggered surface properties of a glycolized PolyEthylene Terephthalate film by surface-initiated ATRP of azobenzene monomer[END_REF] RAFT [START_REF] Zhou | Photoswitching of glass transition temperatures of azobenzene-containing polymers induces reversible solid-to-liquid transitions[END_REF] or Cu(0)-RDRP [START_REF] Zhang | Long Alkyl Side Chains Simultaneously Improve Mechanical Robustness and Healing Ability of a Photoswitchable Polymer[END_REF] are used to polymerize azobenzenes bearing an acrylate or methacrylate as polymerizable group. RAFT polymerization can have a disadvantage for a photoisomerization study because usually a sulfur is present on the control agent and bring a yellow color to the final polymer, i.e. meaning an absorption in the UV-visible wavelength range. In ATRP polymerization, copper and an amine-based ligand are generally used as the catalyst. The removal of green-coloured copper bromide from the final polymer has to be done as previously described for coloured RAFT agent and is moreover tedious and time/cost consuming. So finally, NMP Polymerization reaction was chosen as simple to enforce experimentally, with only two species present in the reactor, the initiator and the monomer. To the best of our knowledge, no scientific paper reports the homopolymerization of azobenzene monomers by Nitroxide-Mediated Polymerization (NMP) [START_REF] Nicolas | Nitroxide-mediated polymerization[END_REF] using an alkoxyamine (BlocBuilder®) as initiator. Only copolymerization with azobenzene in a small amount were described. Yoshida et al. [START_REF] Yoshida | Preparation of micelles with azobenzene at their coronas or cores from 'nonamphiphilic' diblock copolymers[END_REF] used NMP to copolymerize only 1 M% of styrenic azobenzene with BPO as initiator and 4-methoxy-TEMPO as a counter-radical. Srichan et al. [START_REF] Srichan | Influence of strong electron-donor monomers in sequencecontrolled polymerizations[END_REF] reported the copolymerization by NMP initiated by the BlocBuilder®, of a styrenic monomer with an initial 5 mol% N-substituted maleimide presenting an azobenzene substituent.

Herein, a series of azobenzene homopolymers with four different functional groups were synthetized by NMP to control the polymer chain length and dispersity, and finally study their influence on the RSLPT. We investigated the effect of alkyl-and alkoxy-based electron-donor groups, linkers length and polymer chain lengths on the photo-physical properties of the prepared azopolymers. Structure of monomers and polymers were characterized by 1 H NMR spectroscopy and the molecular weight/dispersity values of the polymers determined by size exclusion chromatography (SEC). The photoisomerization behavior in solution was followed by 1 H NMR in CDCl 3 . Finally, the photoinduced liquefaction of the bulky polymers were studied by optical microscopy under UV irradiation.

Results and Discussion

Azomonomer synthesis.

The synthetic methodology for the azomonomers is represented in Scheme 1. The four azobenzene monomers were prepared from the corresponding hydroxyazobenzenes previously synthetized (See supplementary data "part 1"), with high yields (75 -91 %) after purification (Table 4).

Scheme 1. Synthetic methodology for azomonomers preparation.

All monomeric chemical structures obtained were confirmed by 1 H NMR in CDCl 3 (supplementary data Figure S1 to S4 "part 2").

Azopolymerization by NMP.

All polymers were synthetized using NMP carried out at 115 °C. The polymerization was initiated by the BlocBuilder® alkoxyamine under control of the SG1 nitroxide radical. First, a kinetic study of the azomonomer "A" polymerization with a [M]/[I] of 14 and 27 was performed and each point of kinetics in `Figure 1 represents a monomer/initiator mixture splited from a "mother" solution without any purification process, i.e. precipitation (Table S1 in SI "part 2"). The logarithm of the conversion evolves linearly versus the polymerization time (Figure 1E) and the theoretical molecular weights fit well the experimental number-average molecular weights until 80 % conversion (Figure 1C). After 80 % of conversion, the polymerization starts to become less-controlled and the dispersity values increase with a wideness of the molecular weight distribution and shoulder (Figure 1B andD). In the same time, the logarithmic values of the conversion become lower than the theoretical ones (Figure 1E) that means the appearance of dead chains due to termination reaction.

In the literature, few azopolymers with high molecular weight, i.e. > 10-15kD, are obtained. Indeed, Cieciórski et al. [START_REF] Cieciórski | New photoresponsive poly(Meth)acrylates bearing azobenzene moieties obtained via atrp polymerization exhibiting liquid-crystalline behavior[END_REF] tried to optimize the synthesis of homopolymers of azobenzene by ATRP in 2021. Despite all their attempts with prolonged time of polymerization from 24 to 140 h or high M/I value ratio from 50 to 100, a maximum molecular weight was reached around 15 000 g.mol -1 . Wu and coll. [START_REF] Zhou | Photoswitching of glass transition temperatures of azobenzene-containing polymers induces reversible solid-to-liquid transitions[END_REF] synthetized homopolymers of azobenzene by RAFT polymerization (9 900 g.mol -1 , Ð = 1.32) and in order to achieve higher molecular weight, they used a preparative cycling SEC to obtain a molecular weight of 27 000 g.mol -1 with a dispersity value of 1.07. In another work, [START_REF] Zhang | Long Alkyl Side Chains Simultaneously Improve Mechanical Robustness and Healing Ability of a Photoswitchable Polymer[END_REF] they synthesized an azopolymer at 21 300 g.mol -1 with a dispersity of 1.37 by Cu(0)-RDRP. This difficulty to produce a homoazopolymer with a high molecular weight is the most often attributed to a strong steric hindrance or to the propagating radical which could interact with the azo group and then inhibit the polymerization. [START_REF] Wang | Azo Polymer Syntheses[END_REF] In order to analyze the effect of the nature and the size of the azopolymer chain, different kinds of azopolymers were synthesized (Table 1).

Table 1. Experimental conditions and features of Azopolymers.

Name Monomer Polymerization Time (h) [M]/[I] [a] Conv (%) [b]
Mn theo (g.mol -1 ) [c] Mn exp (g.mol -1 ) [d] Ð [e] Tg (°C) [ If we decided to push the conversion, i.e. higher than 90 %, for recovering the largest amount of polymers from the introduced monomers, the final dispersity values remained nevertheless relatively low and comprised between 1.2 to 1.4 due to the precipitation recovery process.

In order to enhance the chain length of the azopolymer the initial M/I value ratio was increased up to 109 (Figure S5 and Table S1 in SI "part 2") but these experiments were unsuccessful.

Another option was to performed the NMP polymerization in DMF under microwave-assisted process (Table 2). [START_REF] Marcasuzaa | Microwave synthesis: An alternative approach to synthesize conducting end-capped polymers[END_REF] Table 2. Polymerization features and characteristics of azopolymer prepared from monomer "A" under microwave irradiation

Name [M]/[I] [a] [M] (mol.L -1 ) Time (min)
Cv [b] Mn theo

[c]
Mn exp

[d]
Ð [e] « However, with this method, conversion was limited (< 59 %) and the highest molecular mass was 6 900 g.mol -1 . The effect of the microwave is still open and the reason for the increase in the polymerization rate (Rp) is under debate. Basically, two alternatives have been proposed: 1) one attributes the increase in Rp to thermal effects, which means rapid heating of the polymerization mixture and formation of super-heated regions. This case can be clearly observed where polymerization rate increases and a number of experimental results clearly demonstrate this scenario [START_REF] Kwak | Critical evaluation of the microwave effect on radical (Co)polymerizations[END_REF][START_REF] Sugihara | Assessment of the influence of microwave irradiation on conventional and RAFT radical polymerization of styrene[END_REF][START_REF] Paulus | High temperature initiator-free RAFT polymerization of methyl methacrylate in a microwave reactor[END_REF].

2) The alternative idea is based on non-thermal effects, i.e. the effect of the microwave irradiation on the kinetic rate coefficients. This scenario is not widely accepted, but there are data in literature [START_REF] Marcasuzaa | Microwave synthesis: An alternative approach to synthesize conducting end-capped polymers[END_REF][START_REF] Brown | Ultra-fast microwave enhanced reversible addition-fragmentation chain transfer (RAFT) polymerization: monomers to polymers in minutes[END_REF][START_REF] Roy | Rapid Block Copolymer Synthesis by Microwave-Assisted RAFT Polymerization[END_REF][START_REF] Rigolini | Microwave-assisted nitroxide-mediated radical polymerization of acrylamide in aqueous solution[END_REF] that are difficult to explain in the absence of non-thermal effects [START_REF] Zetterlund | RAFT Polymerization under Microwave Irradiation: Toward Mechanistic Understanding[END_REF]. In a small number of these cases a non-thermal effect has been claimed [START_REF] Zetterlund | RAFT Polymerization under Microwave Irradiation: Toward Mechanistic Understanding[END_REF].

Independent of the mechanistic reason for the increase of the polymerization rate, the increase can be advantageously used to reduce the process time. Indeed, the polymerization time is highly reduced from hours to minutes. Indeed, we can highlight the experiment "3" where an azopolymer with a molecular weight of 4 400 g.mol -1 with a low dispersity (1.14) is obtained for a reaction time of 15 min, in comparison with 3 h under thermal activation.

2.3

Reversible photoisomerization and stability behavior in solution 2.3.1 Identification of cis and trans isomers.

The isomerization behavior of hydroxy-azobenzene compounds and azopolymers were first studied by 1 H NMR in CDCl 3 .

This technique was chosen instead of UV-visible spectrophotometry because it allows the direct reading of the proportion of the cis/trans forms without calibration steps. For small molecules such as hydroxy-azobenzenes and azomonomers, NMR peaks are well defined (Figure 2). 1 H NMR analysis of azobenzene like molecules in the trans form is common, [START_REF] Zhang | Long Alkyl Side Chains Simultaneously Improve Mechanical Robustness and Healing Ability of a Photoswitchable Polymer[END_REF] and peaks are well attributed. For cis isomers the peak identification is more difficult and scarcely presented in the literature. In order to attribute the 1 H NMR peaks of the cis isomers COSY NMR (Figure S6 in SI "part 2") and HMBC (Figure S7 in SI "part 2") allowing to respectively observe coupling between neighboring protons and between carbon and proton in J2 and J3 were performed to attribute correctly all the observed peaks (Figure 2). Characteristic peaks due to the cis isomer are shielded (7.1, 6.9, 6.8 and 6.7 ppm) by comparison with the trans isomer (7.9, 7.8, 7. Histogram representing the trans half-life time in seconds calculated from a 1 st order kinetic model (B).

In the case of the trans-to-cis isomerization, the kinetic follows as expected a 1 st order law. [START_REF] Bandara | Photoisomerization in different classes of azobenzene[END_REF] As shown by Figure 3, the transto-cis isomerization is fast since the half-life time of the trans isomer varied between 110 and 185 seconds for the irradiation power used in the study. Increase of the chain length from PA1 to PA4 and PB1 to PB2 seems to slow down the speed of the trans-to-cis isomerization. Even if, it is known that the chemical structure seems to have a relative impact on the trans-to-cis isomerization process [START_REF] Schweighauser | Attraction or Repulsion? London Dispersion Forces Control Azobenzene Switches[END_REF][START_REF] García-Amorós | Recent advances towards azobenzene-based lightdriven real-time information-transmitting materials[END_REF] for a similar Mn (PA3, PB1, PC and PD), trans-to-cis isomerization of PB1 and PC is faster. By comparing the chemical structure of these polymers, one can suppose that the presence of longer alkyl chain on the para position of the azo group, i.e. PC compared to PA3, leads to a relative increase of the trans-to-cis isomerization speed. This effect may be due to an increase of the steric effect or an increase of the inductive electron donor capacity. Oppositely the presence of a mesomeric electron-donor group, i.e. ether function, for PD compared to PC, leads to the opposite effect. The increase of the length of the alkoxy-linker, between PB1 and PA3, seems to increase the rate of the trans-to-cis isomerization in solution.

Stability of the cis isomer.

The rate of the thermal cis-to-trans isomerization is known to be determined by the mechanism through which the process takes place (inversion, rotation or mixed mechanism). [START_REF] García-Amorós | Recent advances towards azobenzene-based lightdriven real-time information-transmitting materials[END_REF] As shown in Figure 4, for all polymers the cis-to-trans thermal isomerization followed also a first order kinetic. [START_REF] Bandara | Photoisomerization in different classes of azobenzene[END_REF] Because solution concentration has an important effect on the cis half-life time value (Figure S9 in SI "part 2"), we compared azopolymer solution with the same concentration (8.3 g.l -1 ). The molecular weight has no significant effect on the cis-to-trans kinetic rate in solution in this range of macromolecular length (Figure 4A). As

shown by Figure 4B, the thermal cis-to-trans photo-isomerization rate slowed down upon increasing the hydrophobic chain length (PC). This result is consistent with the results reported by Chen et al. [START_REF] Chen | Synthesis of photo-responsive azobenzene molecules with different hydrophobic chain length for controlling foam stability[END_REF] Increasing the steric hindrance of the alkyl chain We have shown that the photo-isomerization behavior of azopolymers is impacted by the chromophore structure and particularly by the size and the electron-donor capacity of the substitutes.

Photoinduced solid-to-liquid properties of azopolymers.

Under UV irradiation, some azopolymers can demonstrate a solid-to-liquid transition. [START_REF] Zhou | Photoswitching of glass transition temperatures of azobenzene-containing polymers induces reversible solid-to-liquid transitions[END_REF] Table 3 shows the observed solidto-liquid SL transition under an optical microscope of the series of synthesized azopolymers when illuminated at 365 nm with an irradiance of 0.34 W.cm -2 . Since the change of color of the azopolymers, i.e. trans-to-cis isomerization, occurred before the SL transition, we suspect that the Tg cis of our azopolymers are above room temperature and that a supplementary heating is needed to exceed the Tg cis and cause the SL transition. The increase of the material temperature can be provided by increasing the irradiation time. Therefore, the higher the Tg cis of the material, the longer the irradiation time will be. The quantification of the amount of heat that has to be brought to the different azopolymer for the SL transition occurrence is not in the aim of the present study and will be reported in a future publication.

To evaluate the effect of the molecular weight on the SL transition, we can compare PA1 to PA4 and PB1 to PB2 azopolymers. For these polymers, an increase in molecular weight leads to longer irradiation time needed to make the SL transition. As confirmed by the measured value of Tg cis (Table 3) we believe that the observed longer irradiation time observed for the SL transition for azopolymer with higher Mn can be explained by the increase of Tg cis .

Concerning the effect of the molecular composition of the chromophore, if we compare PA3 with PB1, PC and PD with similar molecular weights, we can see that the SL transition is faster for PB1, PC and PD than for PA3. For these polymers either the linker (PB1) or the para-substituent (PC, PD) are longer than those of PA3.

We believe that the resulting enhanced flexibility leads to a decrease of the Tg cis (Table 3) and thus a faster SL transition. Concerning the linker length, our result agrees with those of Wu et al., [START_REF] Weis | Effects of Spacers on Photoinduced Reversible Solid-to-Liquid Transitions of Azobenzene-Containing Polymers[END_REF] and Akiyama et al. [START_REF] Akiyama | Reworkable adhesives composed of photoresponsive azobenzene polymer for glass substrates[END_REF] showing that SL transition at room temperature was only observable with higher alkyl length for azopolymer with a structure similar to that of our PA, PB and PC series. When we compare PC and PD, we can see that the SL transition is faster for PC. Here we believe that the molecular structure of PD leads to a higher rigidity of the polymeric chain and thus a higher Tg cis (i.e. 44°C) compared to PC (i.e. 31°C).

Reversibility of the SL transition.

As shown on Table 3, four days in the dark are needed for all the azopolymers to recover their yellowish color characteristic of the trans form. This result agrees with the good stability of the cis form observed in solution (Figure 4). After 4 days in the dark the azopolymers have also recovered their initial solid property (Figure 6). Atomic Force Microscopy (AFM) force-distance curves representative of the pristine PA3 material (trans form) are characterized by a weak hysteresis between the approach and retraction phases, a low adhesion (i.e. maximum negative force) and high slope in the region of positive indentation (i.e. high rigidity). These curves are characteristic of a solid-like behavior of polymeric material. [START_REF] Delorme | Experimental evidence of ultrathin polymer film stratification by AFM force spectroscopy[END_REF] In comparison the curves, obtained after 10 min of UV irradiation (i.e. when the SL transition is complete) show a high hysteresis between the approach and retracting curves characteristic of a viscous fluid. [START_REF] Delorme | Experimental evidence of ultrathin polymer film stratification by AFM force spectroscopy[END_REF] Finally, when the liquid-like sample is kept in the dark for four days the force distance curves return to their original shape which demonstrate the reversibility of the SL transition.

Conclusion

In this study, in order to understand the structure-property relationship on photoinduced solid-to-liquid transition properties, Nitroxide Mediated Polymerization was used to synthesize well-defined azopolymers. Four acrylate monomers (A, B, C and D) were designed and then polymerized. Four different molecular weight, i.e. from 3 400 to 8 100 g.mol -1 were targeted for the "A" monomer C 6 H 12 -azo-CH 3 . Higher molecular weights are not reachable, even under micro-assisted polymerization, because of the steric hindrance of the monomer. Photoisomerization properties and photo-reversible solid-to-liquid transitions were studied as function of the molecular weight and the molecular structure of the azopolymers. Both have an impact on the features of the reversible solid-to-liquid transition. In particular, the higher the length of the substituent or the linker, the faster the transition. This is due to a gain in flexibility of the side chain that leads to a decrease in the Tg value of the cis form. These results have to be considered in the design of novel azopolymers for practical applications.

Experimental Section

Materials

Triethylamine (99 %, ABCR), 4-Dimethylaminopyridine (≥ 99 %, DMAP, Sigma-Aldrich), Acrylic Anhydride (> 95 %, TCI) were used without purification. The BlocBuilder® alkoxyamine (2-methylaminoxypropionic-SG1) used as the initiator was supplied by Arkema. All solvents and Dialysis Membrane (Spectra/Por® 6 prewetted RC Tubing, MWCO: 2 kD) were purchase from VWR and used as received. Dry THF was dried using a PureSolv (INERT) solvent purification system under N 2 .

Synthesis

Synthesis of hydroxy-azobenzene. Four different hydroxy-azobenzenes (Figure 7) were synthetized and characterized following the method described in supplementary data "Part 1". Synthesis of azomonomer. All azomonomers were prepared using the same method (Table 4). Hydroxyazobenzene (1 eq), anhydride acrylic (1.5 eq), triethylamine (1.2 eq), 4-dimethylaminopyridine (0.3 eq) are mixed in dry THF and degassed during 15 min. The reactional mixture was heated at 40°C during 24 h. To purify the monomer, the solution is filtered and then washed by precipitation in water (2 times). To finish, the powder is dried under vacuum at 30°C. Azomonomers were characterized by 1 H NMR in CDCl 3 (supplementary data Figure S1 to S4 "part 2"). Then, the mixture is exposed to microwave at 300 W using the SPS mode with a limit of temperature at 115 ± 1°C. After the exposition, 1 H NMR and SEC analysis were realized. In this case, the conversion was 59 % and the final number-average molecular weight was 3 700 g.mol -1 with a dispersity of 1.18.

Photo-isomerization experiments. Irradiation of solutions and powder were performed at 365 nm with a High-Power LED from ThorLabs (SOLIS-365C). Irradiance was controlled at 0.10 W.cm -² and 0.34 W.cm -² for solution and powder respectively.

Characterization

The molecular weight and dispersity values of polymers were measured using size exclusion chromatography (SEC) using THF as eluent (flow rate 1.0 mL.min UV-visible absorbance of polymers was measured on a double-beam spectrophotometer (Cary 5000, Varian).

Spectra were recorded at room temperature in the range 250-700 nm.

Glass transition temperature (Tg) were measured with Differential scanning calorimetry (DSC) METTLER TOLEDO DSC 3 at 20°C.min -1 . Two cycles of temperature were realized to -10°C to 150°C to -10°C. The value of the Tg of the second cycle was chosen for the Tg of the trans isomer. After these two cycles, samples were irradiated 30 min under 365 nm and then analyzed again between -10°C to 150°C at 20°C.min -1 in order to obtain the Tg of the cis isomer. The STAR e software was using for DSC treatment.

The irradiation time was measured as the time from which the liquefaction of the powder is observed by optical microscopy (x20 objective) when the material is subjected to irradiation at 100 mW/cm².

Figure 1 .

 1 Figure 1. Chromatography trace of the kinetic [M]/[I] of 14 (A) and [M]/[I] of 27 (B) of the azomonomer "A"; Number-average molecular weight versus conversion, solid line represents the theoretical molecular weight (C); Dispersity versus conversion (D) and logarithmic of conversion against time of polymerization (E); circle blue: [M]/[I] of 14 and triangle grey: [M]/[I] of 27.

  3 and 7.0 ppm), each peak representing 2 protons due to the symmetry.As usual feature of the polymeric state, for the azopolymers, assigned peaks are widened. Apart from this broadening, the chemical shifts of the aromatic protons of the azopolymers follow the same trend as their precursor's counterparts. Due to the nearly symmetric nature of species "d and PD", the 1 H NMR attribution is different and represented in supplementary data (figure S8 in SI "part 2").

Figure 2 .

 2 Figure 2. Aromatic area of 1 H NMR spectra of the cis and trans isomers of hydroxyazobenzene "a" and azopolymer "PA2".

Figure 3 .

 3 Figure 3. Percentage of trans isomer of the different azopolymers as function of the time under irradiation at 365 nm (A).

Figure 4 .

 4 Figure 4. Evolution of the cis isomer percentage of azopolymers as function of the time in the dark regarding (A) the effect of the molecular weight with PA1, PA2, PA3 and PA4 (A) and (B) the effect of the nature of the azobenzene with PA3, PB1, PC and PD. Azopolymer (Azopolymer are initially irradiated at 365 nm to obtained the cis isomer. Inset represents the cis-half time in hour of the cis isomer calculated with a 1 st order kinetic model).

For

  all the azopolymers, we can observe a two-step mechanism for the SL transition. At first, for short irradiation time, i.e. t = 15 s, a transition from yellowish to reddish color is observed without apparition of a liquid phase. This change of color can be attributed to the trans-to-cis isomerization of the azopolymer. Indeed, as shown in Figure5, upon UV irradiation, the π-π* absorption band of trans azobenzene groups at ≈ 350 nm decreased. Correspondingly, the n-π* absorption band of the cis isomer at ≈ 450 nm increased with the irradiation time. The fast trans-to-cis isomerization observed in the solid phase confirms our results obtained in solution. In a second step, with prolonged irradiation, all the synthetized polymers show an SL transition at an irradiation time depending of the polymer nature. At present, for azopolymers the SL transition under irradiation is explained by the fact that the trans-to-cis isomerization decreases the glass transition temperature Tg of the material.[START_REF] Weis | Effects of Spacers on Photoinduced Reversible Solid-to-Liquid Transitions of Azobenzene-Containing Polymers[END_REF]

Figure 5 .

 5 Figure 5. Representative UV-visible spectra of cis and trans forms for azopolymers (herein PA2).

Figure 6 .

 6 Figure 6. Representative AFM force-distance curve on PA3 before and after irradiation, and for 4 days in the dark. Arrows indicate the direction of the AFM scanner toward the sample (i.e. approach and retraction)

Figure 7 .

 7 Figure 7. Denominations of the hydroxy-azobenzene precursors synthetized: (a) HO-C 6 H 12 -azo-CH 3 , (b) HO-C 10 H 20 -azo-CH 3 , (c) OH-C 6 H 12 -azo-C 6 H 13 and (d) HO-C 6 H 12 -azo-OC 6 H 13 .

- 1 )

 1 at 35°C. SEC is equipped with a Viscotek VE 5200 automatic injector, a pre-column and two columns (Styragels HR 5E and 4E (7.8´ 300 mm), an orbit wyatt and 4 detectors: UV-visible spectrophotometer (Viscotek VE 3210), a Multi-angle Light Scattering detector (Wyatt Heleos II), a viscosimeter (Viscostar Neon Wyatt) and a refractive index detector (Viscotek VE 3580). Numberaverage molar mass (Mn) and dispersity (Mw/Mn, where Mw is the mass-average molar mass) of the polymers were calculated from a calibration derived from polystyrene standards. The polymer samples were prepared at 5 g.L -1 and filtered through 0.45 μm PTFE filters.1 H NMR, COSY and HMBC experiments were carried out on a Bruker AVANCE 400 MHz spectrometer in CDCl 3 at 25°C. Chemical shifts were reported as ppm downfield from TetraMethyl Silane (TMS). The polymer solution in CDCl 3 were prepared at 8.3 g.L -1 .

Table 3 .

 3 Optical microscopy images (350x270 µm²) and irradiation time of the reversible SL transition and glass transition temperature measured by DSC of the cis isomer for the synthetized azopolymers

	Azopolymer	Pristine trans form	15 s of irradiation (365 nm -0.34W.cm -2 )	Liquid phase (irradiation 365nm t>120s)	SL transition time irradiation	Tg cis [°C]	After 4 days in the dark
	PA1				30 s	31
	PA2				45 s	45
	PA3				45 s	44
	PA4				45 s	42
	PB1				30 s	38
	PB2				60 s	50
	PC				15 s	31
	PD				30 s	44

Table 4 .

 4 Experimental conditions for azomonomer synthesis.Synthesis of azopolymer by NMP using microwave reactor. Experimental conditions for entry 2 in table 2with an initial [M]/[I] equal to 27 is given as an example. 60 mg of azomonomer A (366 g.mol -1 , 164 µmol) and 2.3 mg of BlocBuilder® (381 g.mol -1 , 6 µmol) were mixed in 0.25 mL of DMF and degassed during 10 min.

	AzoMonomer	M OH-azo (g.mol -1 ) [a]	[OH-azo] (mol.L -1 )	n OH-azo (mmol)	% Yield [b]
	A: C 6 H 12 O-azo-CH 3	312	3.2 x 10 -4	12.8	90
	B: C 10 H 20 O-azo-CH 3	368	2.7 x 10 -4	2.72	91
	C: C 6 H 12 O-azo-C 6 H 13	382	2.6 x 10 -4	7.9	75
	D: C 10 H 20 O-azo-OC 6 H 13	398	2.6 x 10 -4	7.6	82
	[a] OH-azo = hydroxyazobenzene, [b] Yield after purification.
	Synthesis of azopolymer by NMP. Azopolymers were synthetized using the same protocol. For example, to
	obtain an azopolymer PA3 with an initial [M]/[I] equal to 27, 0.5 g of azomonomer C 6 H 12 -azo-CH 3 (366 g.mol -1 ,
	1.36 mmol) and 19.3 mg of BlocBuilder® (381 g.mol -1 , 0.05 mmol) were mixed, degassed during 10 min and
	heating at 115°C during 16 h. The final solution was analyzed by 1 H NMR before purification, in this case 95 %
	of conversion were obtained. After purification by dialysis in a mixture of THF, ethanol and water, the purified
	sample is analyzed by

1

H NMR and SEC in THF. The obtained number-average molecular weight was 8 100 g.mol -1 with a dispersity of 1.27.

Acknowledgements

We gratefully acknowledge Brewen le Floch for the synthesis of hydroxy-azobenzene, Sullivan Bricaud and Abdel Khoukh for COSY and HMBC NMR analyses, and Alexandre Bénard for the HRMS analyses.

The ANR-20-CE06-0014-01 (Photoprint) for funding.

Data availability statement

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

CRediT authorship contribution statement

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Arancha Hevia

Patricia Ruas-Madiedo

Miguel Angelo Faria

Valérie Petit

Bruna Alves

Paula Alvito

Elena Arranz

Shanna Bastiaan-Net

Milena Corredig

Wieneke Dijk

Didier Dupont

Linda Giblin

Brigitte Anna Graf

Alina Kondrashina

Helena Ramos

Lorena Ruiz

Marta Santos-Hernández

Laura Soriano-Romaní

Lidia Tomás-Cobos

Santiaga María Vivanco-Maroto

Isidra Recio

Beatriz Miralles

A Shared Perspective on in Vitro and in Vivo in Models to Assay Intestinal Transepithelial Transport of Food Compounds

Keywords: intestines, transepithelial transport, nutrient bioavailability, food compounds

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

INTRODUCTION

To assess the impact on human health, it is necessary to evaluate the bioaccessibility and bioavailability of compounds in food such as bioactives, macro-and micronutrients, allergens, and contaminants with either positive or negative effects on health. This information underpins the cause-andeffect relationship of bioactive compounds, identifies the minimum intake of essential micronutrients, and establishes safe doses of contaminants or allergens. The models used for this purpose typically adhere to the general guidelines used in assessing drug bioavailability. Absorption, distribution, metabolism, and excretion (ADME) (with potential toxicity) are the key biological processes involved in drug, nutrient, and molecule's pharmacokinetics. These compounds should escape intestinal metabolism in the lumen and gut wall, in order to pass through the epithelial cell layer, to enter the portal vein, and to reach the liver and ultimately the systemic circulation. [START_REF] O'shea | Best Practices in Current Models Mimicking Drug Permeability in the Gastrointestinal Tract -An UNGAP Review[END_REF] It has also been shown that food-derived substances and the structures formed during digestion are able to modulate the intestinal absorption functions by themselves, [START_REF] Shimizu | Modulation of Intestinal Functions by Food Substances[END_REF] which adds an extra complexity to the food-intestinal epithelium-absorption equation. The food matrix will also contribute to or undermine compound survival. Along with studies in humans, digestion and absorption models are needed to understand the mechanisms beyond oral delivery, making it possible to systematically study an increasing number of molecules of interest and their interactions. The present work aims to put into perspective the wide-range of models used to assay the permeability of food compounds by taking into consideration the particular nature of the different molecules, and, where possible, comparison with in vivo data. This work relies on the experience of a consortium of researchers with activities focused on intestinal absorption models.

IMPACT OF THE PROPERTIES OF FOOD-DERIVED COMPOUNDS ON THEIR TRANSPORT THROUGH THE INTESTINAL EPITHELIUM

The intrinsic characteristics of the ingested compounds and the actual form in which they reach the intestinal epithelium determine the mechanism to be absorbed, and the transport model should ideally mimic this. Figure 1 outlines the major transport mechanisms for macronutrients.

Transport of Lipids.

Dietary lipids consist of a wide array of chemically diverse polar and nonpolar lipids. Triacylglycerols (TAG) are the most abundant lipids, accounting for 90-95% of the total energy provided by dietary fat, which also includes phospholipids, sterols (e.g., cholesterol and β-sitosterol), and other compounds like lipophilic vitamins. Pancreatic lipases in the small intestine hydrolyze TAG into diacylglycerols, monoacylglycerols, and free fatty acids (FFAs). FFAs are then incorporated into spontaneously forming mixed vesicles (or dietary mixed vesicles), which shuttle lipophilic compounds through the "unstirred water layer" to reach the enterocyte brush border. At the brush border, the mixed vesicles disperse and unesterified FFAs cross passively through the membrane into enterocytes, where they are immediately re-esterified to form TAGs. TAGs are then packaged into chylomicrons, which are initially secreted into intestinal lymph vessels as they are too large for blood capillaries. Long chain FFAs can be actively taken up by enterocytes and are also esterified into TAGs before incorporation into chylomicrons, while short chain FFA are sufficiently hydrophilic to be transported directly to the blood circulation. [START_REF] Mekki | Butter Differs from Olive Oil and Sunflower Oil in Its Effects on Postprandial Lipemia and Triacylglycerol-Rich Lipoproteins after Single Mixed Meals in Healthy Young Men[END_REF] Intracellular assembly of TAG-rich chylomicrons requires predominantly apolipoprotein B-48 biosynthesis and subsequent cotranslational lipidation with TAGs. [START_REF] Black | Development and Physiological Regulation of Intestinal Lipid Absorption. I. Development of Intestinal Lipid Absorption: Cellular Events in Chylomicron Assembly and Secretion[END_REF] Lipid bioavailability in humans is dependent on the initial emulsification (and thus droplet size) of dietary lipids to allow for lipase to digest TAGs into FFAs and the subsequent incorporation into mixed vesicles who shuttle digested lipids to the enterocyte brush border.

The intestinal transport of lipids in vitro necessitates the prior formation of mixed micelles, a step accomplished through relevant gastrointestinal simulations such as the INFOGEST method. However, when conducting studies directly involving lipids, it is imperative to prepare the mixed micelles beforehand. At the cellular level, the most crucial factor of an in vitro model for lipids is their ability to form and secrete lipoproteins. In addition, the expression of fatty acid binding proteins is relevant to enterocytes functionality in studies involved in lipid transport and metabolism. The human immortalized Caco-2 cell line, once polarized and differentiated to monolayers, is well capable of lipoprotein production and secretion, [START_REF] Nauli | Chylomicrons Produced by Caco-2 Cells Contained ApoB-48 with Diameter of 80-200 Nm[END_REF] which are the largest and most efficient lipoprotein lipid transporters. Caco-2 cells have been compared to human colonic explants for their ability to esterify lipids, synthesize apolipoproteins, and assemble lipoproteins. [START_REF] Levy | Caco-2 Cells and Human Fetal Colon: A Comparative Analysis of Their Lipid Transport[END_REF] Differentiated Caco-2 cells, like enterocytes, incorporate oleic acid into cellular phospholipids, TAGs and cholesterol esters, however with limited capacity to export newly synthesized lipids. Depriving Caco-2 cells of fetal bovine serum in the apical medium compartment during differentiation for one to two weeks prior to oleic acid treatment, was found to improve chylomicron production. [START_REF] Tomassen | Dietary Fat Induced Chylomicron-Mediated LPS Translocation in a Bicameral Caco-2cell Model[END_REF] Beyond the standard Caco-2 models, cocultures with mucus secreting cells, Caco-2/ HT29-MTX, demonstrate a higher functionality for oleic acid uptake, storage, and subsequent basal lipid release versus monoculture Caco-2 barriers. [START_REF] Berger | Adipocytes as Lipid Sensors of Oleic Acid Transport through a Functional Caco-2/HT29-MTX Intestinal Barrier[END_REF] Salvini et al. have shown that Caco-2/TC7 cells (a subclone of Caco-2) exhibit a higher rate of secretion of oleic acid (18:1) than the parental Caco-2 cells, with 86% of net apical uptake which compared well to 97% observed in human subjects. [START_REF] Salvini | Functional Characterization of Three Clones of the Human Intestinal Caco-2 Cell Line for Dietary Lipid Processing[END_REF] 2.2. Transport of Amino Acids, Peptides, and Proteins. In the healthy gut, dietary proteins are hydrolyzed to a different extent into peptides and amino acids according to their natural or processing-derived susceptibility to proteases. It is classically accepted that 90% of absorbed dietary proteins are represented in the circulation by amino acids and 10% as dipeptides and tripeptides. However, food-derived peptides over this number of amino acids have been also identified in plasma in animals and in humans following dedicated extraction and up-to-date peptidomic tools. [START_REF] Sánchez-Rivera | Bioavailability and Kinetics of the Antihypertensive Casein-Derived Peptide HLPLP in Rats[END_REF][START_REF] Caira | In Vivo Absorptomics: Identification of Bovine Milk-Derived Peptides in Human Plasma after Milk Intake[END_REF] In the human gut, amino acids have a number of transport systems involved in the absorption from the lumen into the enterocytes that vary in solute specificity and may present an electroneutral or electrogenic transport process. These include b 0,+ AT, rBAT, CAT1, CAT2, y + LAT1, and y + LAT2 for basic amino acids, B 0 AT and LAT1 for neutral amino acids, and EAAT1 and EAAT3 for anionic amino acids. [START_REF] Gilbert | REVIEW: Peptide Absorption and Utilization: Implications for Animal Nutrition and Health[END_REF] The basolateral membrane of the enterocytes also expresses at least six different Na +dependent and -independent amino acid transport systems that mediate the exit to the blood. These latter transport systems may work in either direction depending on the luminal amino acid concentration and cellular demand. Quantitatively, the absorption of peptides across the intestinal lumen is the predominant mode of absorption of protein digestion products. Peptides in the intestinal lumen can be transported through the intestinal epithelium into the bloodstream via a paracelullar route, transcytosis, passive transcellular diffusion, and/or peptide transporters. Di-and tripeptides are efficiently transported by proton gradient cotransport. The solute carrier 15 (SLC15) family of transporters are H + -dependent transporters involved in the uptake of di-and tripeptides in the cells. Within this family PEPT1 (SLC15A1) and PEPT2 (SLC15A2) mediate intestinal absorption of di-and tripeptides, including anionic and cationic peptides, as well as different peptide drugs. In PEPT1, transport efficiency depends on the net charge of the peptides and the relationship between membrane potential and external pH, with the transport of neutral and negatively charged dipeptides being stimulated by lowering luminal pH, while an increasing pH is coupled with transport of positively charged peptides. [START_REF] Smith | Proton-Coupled Oligopeptide Transporter Family SLC15: Physiological, Pharmacological and Pathological Implications[END_REF] Other authors have reported that PEPT1 is unable to bind peptides with high positive charge, such as KK or KWK. [START_REF] Xu | Bioavailability of Bioactive Peptides Derived from Food Proteins across the Intestinal Epithelial Membrane: A Review[END_REF] In addition to peptide transport, several human studies have confirmed that small amounts of intact proteins, such as cow's milk β-lactoglobulin and hen's egg ovalbumin, can be found in the plasma of human volunteers following their consumption. [START_REF] Bøgh | Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?[END_REF] The mechanisms by which these proteins with allergenic potential are transported across the intestinal epithelial barrier include paracellular or transcellular transport. These transport mechanisms depend on a number of factors, including intrinsic protein properties, the physiological state of the intestine, as well as extrinsic, food-related factors. [START_REF] Samadi | The Role of Gastrointestinal Permeability in Food Allergy[END_REF] Moreover, although they have been always thought to be purely secretory cells, small intestinal globlet cells can take up luminal antigen and deliver it to lamina propria dendritic cells. [START_REF] Mcdole | Goblet Cells Deliver Luminal Antigen to CD103+ Dendritic Cells in the Small Intestine[END_REF] LAT1 and LAT2 transporter transcripts and proteins are present in Caco-2, with LAT2 playing an important role in amino acid transport. In contrast with B 0 and y+ systems, which have been reported to be downregulated when Caco-2 cells stop proliferating, L-glutamate transport capacity was found to increase in differentiated cells compared to that found during proliferation, and this increase was correlated with the level of EAAT1 mRNA. [START_REF] Mordrelle | EAAT1 Is Involved in Transport Ofl-Glutamate during Differentiation of the Caco-2 Cell Line[END_REF] Caco-2 cell monolayers express apical and basolateral peptide transporters of the SLC15 and cadherin gene families. Although the Caco-2 model has been proven to be versatile for peptides, the gene expression of SLC15A1 (PEPT1), SLC15A3 (PHT2), SLC15A4 (PHT1), and CDH17 (human peptide transporter 1, HPT-1) has been described to be increased compared to the human gastrointestinal tract, [START_REF] Herrera-Ruiz | Spatial Expression Patterns of Peptide Transporters in the Human and Rat Gastrointestinal Tracts, Caco-2 in Vitro Cell Culture Model, and Multiple Human Tissues[END_REF] which may overestimate in vitro results. However, culture length, cell density, or cell supplier may influence peptide transporter expression levels as it has been evidenced in interlaboratory comparisons. The effect of culture conditions has also been evidenced in HT-29 cells, where culturing in galactose-rich medium favored the expression of peptide transporters PEPT1, PEPT2, PHT1, and PHT2. [START_REF] Jochems | Evaluating Human Intestinal Cell Lines for Studying Dietary Protein Absorption[END_REF] Although HT-29/MTX cells are capable of transporting phenylalanine at a lower rate than Caco-2, and the dipeptide (Gly-Pro) at rates similar to that of Caco-2, the transporters need to be specified.

To elucidate the mechanisms of allergen transport, studies have heavily relied on human intestinal transport models, with the Caco-2 model being the most frequently used. For example, the Caco-2 model was used to show that some, but not all allergens (e.g., wheat α-gliadins, peanut allergens, and the shrimp allergen Pen j 1), increase the Caco-2 monolayer permeability and promote their own paracellular passage. [START_REF] Price | Identifying Epithelial Endocytotic Mechanisms of the Peanut Allergens Ara h 1 and Ara h 2[END_REF] Similarly, the Caco-2 model was used to demonstrate that some allergens, including cow's milk β-lactoglobulin or peanut Ara h 1 and Ara h 2, are transported across the intestinal epithelial barrier by a transcellular route during which they are partially degraded. [START_REF] Price | Identifying Epithelial Endocytotic Mechanisms of the Peanut Allergens Ara h 1 and Ara h 2[END_REF][START_REF] Rytkönen | Enterocyte and M-Cell Transport of Native and Heat-Denatured Bovine β-Lactoglobulin: Significance of Heat Denaturation[END_REF] An in vivo matured, intestinal organoidbased model of human induced pluripotent stem cell (iPSC) was used to demonstrate that cow milk allergens could be transported through goblet cells and secretory antigen passages in humans. [START_REF] Noah | IL-13-Induced Intestinal Secretory Epithelial Cell Antigen Passages Are Required for IgE-Mediated Food-Induced Anaphylaxis[END_REF] The relevance of these mechanistic data for protein and oligopeptide absorption in vivo remains to be confirmed using human studies, where possible, preferably by comparing nonallergic and allergic individuals.

Transport of Carbohydrates.

Most consumed carbohydrates in adulthood are sugars and starches. Sugars include monosaccharides (e.g., glucose, galactose, and fructose) and disaccharides (e.g., lactose, sucrose, and others) which are hydrolyzed by the sucrose-isomaltase and maltaseglucoamylase brush border enzymes within the small intestine. Salivary and pancreatic amylases are responsible for the initial breaking down of nonresistant starch to oligosaccharides, followed by further digestion by small intestinal brush border enzymes (i.e., sucrase, sucrase-isomaltase complex, β-glycosidase complex, trehalase), [START_REF] Mamone | Optimized Extraction and Large-Scale Proteomics of Pig Jejunum Brush Border Membranes for Use in in Vitro Digestion Models[END_REF] which are responsible for the final cleavage into individual sugars. In the small intestine, the monosaccharides are then taken up by the enterocytes through specific transporters. Sodium-glucose linked transporters, SGLT1, are mainly expressed in the upper third of the small intestinal villi, driving the transport of the monosaccharides D- glucose and D-galactose. SGLT1 uses the sodium (Na + ) gradient created by the basolateral Na + /K + -ATPases to take up the monosaccharides into the enterocytes. While SGLT1 is localized at the brush border membrane of the enterocytes, glucose transporter 2 (GLUT2) is thought to be localized to the basolateral membrane and only recruited into the apical membrane under high luminal glucose presence. [START_REF] Kiela | Physiology of Intestinal Absorption and Secretion[END_REF] Na +independent GLUT mediates passive transport, such as fructose transport by the uniporter GLUT5. It is currently accepted that also polysaccharides such as hyaluronan, glucan, or pectin can be absorbed from the intestine by active transport and passive diffusion processes. However, although they have been detected in the intestine, they do not easily reach the systemic circulation, as it was shown for glucan and pectin. [START_REF] Zheng | Advances in Oral Absorption of Polysaccharides: Mechanism, Affecting Factors, and Improvement Strategies[END_REF] Charge, molecular weight, spatial structure, and dose are the main determinants of the transported amount. In addition, they can be taken up by immunosurveillant microfold (M) cells and then distributed to the surrounding mucosal immune system in Peyer's patches.

Studies showed that SGLT1 has a lower level of expression in Caco-2 cells compared to human enterocytes, and this varies between laboratories [START_REF] Hayeshi | Comparison of Drug Transporter Gene Expression and Functionality in Caco-2 Cells from 10 Different Laboratories[END_REF] rendering it difficult to compare with in vivo studies. On the other hand, the presence of GLUT2 in the apical membrane of the differentiated enterocytes and its contribution to glucose absorption, is controversial. This has led to some authors to consider the Caco-2 cell monolayers as a poor model for studying intestinal glucose transport, at least the SGLT1-mediated transport. [START_REF] Kulkarni | Characterization of SGLT1-Mediated Glucose Transport in Caco-2 Cell Monolayers, and Absence of Its Regulation by Sugar or Epinephrine[END_REF] 2.4. Transport of Micronutrients. It has been long believed that intestinal absorption of vitamins and minerals was mostly a passive process, and lipophilic vitamins were absorbed together with digested lipids and hydrophilic vitamins with water. However, carrier mediated absorption is now well established for most hydrophilic vitamins (ascorbate, biotin, folate, niacin, pantothenic acid, pyridoxine, riboflavin, and thiamin). [START_REF] Said | Intestinal Absorption of Water-Soluble Vitamins in Health and Disease[END_REF] Similarly, many minerals are absorbed by the involvement of carrier proteins or specific receptors to control uptake and avoid cytotoxicity (e.g., haem iron). Lipophilic micronutrients are best absorbed when lipids are coingested to trigger a postprandial response; however, there is uncertainty as to what lipid dose is optimal. Carrier systems for the absorption of vitamins A, E, K1, and carotenoids have also been identified while vitamins D and K2 seem to be absorbed via a passive mechanism. [START_REF] Amiot-Carlin | Digestion and Absorption of Lipophilic Food Micronutrients[END_REF] The bioacessibility of lipophilic vitamins in different types of foods is commonly measured by their ability to assemble within micelles during digestion experiments. It is assumed that once they are in the micelles, they will be absorbed. [START_REF] Huey | Bioaccessibility and Bioavailability of Biofortified Food and Food Products: Current Evidence[END_REF] 2.5. Transport of Polyphenols. Polyphenols are secondary metabolites of plants, with the natural biological function to protect against external factors such as the sun′s rays or the infection by microorganisms. The bioavailability of polyphenols is low. [START_REF] Klinder | Impact of Increasing Fruit and Vegetables and Flavonoid Intake on the Human Gut Microbiota[END_REF] Although the aglycones can be absorbed from the small intestine, most dietary polyphenols are in the form of esters, glycosides, or polymers that cannot be directly absorbed. These must be hydrolyzed by intestinal enzymes or by the colonic microbiota, with, for example, procyanidin monomers, dimers, and rarely trimers detected in the plasma. Polyphenols are metabolized and conjugated in the intestinal cells and later in the liver by methylation, sulfation, and/or glucuronidation, or return to the digestive tract through the enterohepatic circulation. In the large intestine, the intestinal nonabsorbed polyphenols and conjugated polyphenols are catabolized to ring-fission products (i.e., phenolic acids) by the enterobacteria. These metabolites can then in turn impact the intestinal immune function. [START_REF] Kawabata | Role of Intestinal Microbiota in the Bioavailability and Physiological Functions of Dietary Polyphenols[END_REF] Hence, the biotransformation of polyphenols and the generated metabolites is a key point in studying the bioavailability. In fact, it has been pointed out that studies on the mechanisms of the biological effects of polyphenols on human cell lines should use the actual metabolites produced in vivo, that is the conjugates of the original aglycones or gut microbiota metabolites. In vitro studies have some limitations on reproducing the human biotransformation enzymes. Janssen et al. [START_REF] Janssen | Cytochrome P450 Expression, Induction and Activity in Human Induced Pluripotent Stem Cell-Derived Intestinal Organoids and Comparison with Primary Human Intestinal Epithelial Cells and Caco-2 Cells[END_REF] compared the expression of cytochrome P450 enzymes in Caco-2 monolayers with human intestinal organoids derived from human iPSC. Caco-2 showed lower gene expression and activity of CYP3A4 than organoids, but a better expression of other enzymes such as CYP1B1, CES1, SULT2A1, and UGT1A8. Still, organoids are considered to be preferable for biotransformation studies.

To impart their biological activity, it is important that these phenolic compounds be released from the food matrix and become bioaccessible. Of particular interest is the effect of processing modifications on the bioavailability of these compounds, and many works have dealt with the means to improve their bioaccessibility, for example, by using vesicles as delivery systems. [START_REF] Sabouri | Sodium Caseinate Stabilized Emulsions as a Delivery System for Epigallocatechin-Gallate: Bioaccessibility, Anti-Proliferative Activity and Intestinal Absorption[END_REF] In the analysis of six dietary polyphenols (caffeic acid, chrysin, gallic acid, quercetin, resveratrol, and rutin), strong correlations have been shown between the experimental partition coefficient and both parallel artificial membrane permeability assays (PAMPA) and Caco-2 cell monolayer permeation data. [START_REF] Rastogi | Evaluation of Physicochemical Properties and Intestinal Permeability of Six Dietary Polyphenols in Human Intestinal Colon Adenocarcinoma Caco-2 Cells[END_REF] The results showed that passive diffusion is the main transport mechanism for these compounds, and their poor absorption is consistent with the documented poor oral bioavailability in humans.

2.6. Transport of Food Contaminants. The European Food Safety Agency, EFSA, has assessed and classified chemicals that can contaminate food into several groups, including natural toxins like mycotoxins, alkaloids and bacterial toxins, environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs), pesticides, polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), metals, and process contaminants such as acrylamide, chloropropanols, and heterocyclic aromatic amines (HAA) (https://www.efsa.europa.eu/en/topics/topic/chemicalcontaminants-food-feed). Moreover, new concerns are emerging, such as microplastics contaminating food. Little information is available on the pharmacokinetics of microplastics, which are prone to produce secondary products and can appear as particles of different sizes. [START_REF] Prata | Microplastics and Human Health: Integrating Pharmacokinetics[END_REF] The gastrointestinal mucosa is the first barrier that these compounds encounter before entering the systemic circulation. If bioaccessible, most of these chemical groups are transported through the transcellular pathway and less through the paracellular way. The transcellular barrier is composed of the surface cell membranes, which has a much larger surface area than the surface of tight junctions (99.9 versus 0.01%), where paracellular transport occurs. [START_REF] Van Breemen | Caco-2 Cell Permeability Assays to Measure Drug Absorption[END_REF] Therefore, compounds using the transcellular pathway (e.g., PAHs, PCBs, pesticides, HAAs, heavy metals) tend to have a higher absorption area available than compounds that cross the epithelium via the paracellular pathway (e.g., some mycotoxins and chloropropanols). However, the absorption pathway alone does not determine the compound levels and bioavailability. The measurement of apparent permeability (P app ), which quantifies the flux of a compound through a membrane while normalizing for the membrane surface area and donor concentration, serves as an objective method for expressing the absorption of toxicants. High permeability is indicated by values greater than 1 × 10 -6 cm/s. This threshold is exceeded in the case of mycotoxins, with compounds as Aflatoxin B1, showing remarkable absorption (78 × 10 -6 cm/s) [START_REF] Sobral | Transport of Mycotoxins across Human Gastric NCI-N87 and Intestinal Caco-2 cell Models[END_REF] or chloropropanols (30 × 10 -6 cm/s) where free but not esterified forms are able to cross the monolayer. [START_REF] Araujo | Assessment of Intestinal Absorption/Metabolism of 3-Chloro-1,2-Propanediol (3-MCPD) and Three 3-MCPD Monoesters by Caco-2 Cells[END_REF] Unfortunately, with a few exceptions, these values are largely absent from the existing literature, probably due to the high number of sampling settings needed. Consequently, future investigations should focus on generating Papp values for more toxicants.

THE CHALLENGE TO DETERMINE IN VIVO ABSORPTION RATES

There are many challenges in the determination of absolute food compounds bioavailability. To determine nutrient bioavailability in humans, simple measurement of nutrient appearance in plasma is not sufficient as many nutrients are tightly regulated and metabolized, and newly absorbed compounds need to be separated from those already present D in the body. This difficulty has been highlighted by in vivo absorption studies with different food components.

The "chylomicron fraction method" separates recently absorbed lipophilic micronutrients. Immediately after absorption in the enterocytes, newly absorbed lipophilic nutrients are indeed "packaged" into chylomicrons, while whole blood contains both previously absorbed and recently absorbed nutrients. Chylomicrons can be isolated from fresh plasma by density gradient ultracentrifugation, and the compound of interest can be then quantified by HPLC or LC-MS, although the method is not valid for hydrophilic micronutrients. Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating dietary fat transported from the intestine in chylomicrons and fat transported from the liver in VLDL by ultracentrifugation. An alternative to the ultracentrifugation is the prediction of chylomicron triglycerides in human plasma using metabolomics tools such as nuclear magnetic resonance and chemometrics. [START_REF] Savorani | High Throughput Prediction of Chylomicron Triglycerides in Human Plasma by Nuclear Magnetic Resonance and Chemometrics[END_REF] Alternatively, chylomicrons may be purified from VLDL in postprandial human plasma samples by an immunoaffinity method. This method improved the evaluation of the relative contribution of the small intestine versus the liver to postprandial lipid fluxes. In addition, to address the dietary influences of postprandial lipids on cardiovascular risk, this method coupled to a tracer feeding study ( 13 C acetate) generated preliminary evidence suggesting enteral conversion of dietary sugar to fat via de novo lipogenesis. [START_REF] Jones | Separation of Postprandial Lipoproteins: Improved Purification of Chylomicrons Using an ApoB100 Immunoaffinity Method[END_REF] These methods, however, result in values of relative bioavailability and not absolute bioavailability. Absolute bioavailability is the total amount of a compound taken up from the gut into the blood and is usually determined in animals as both an oral and an intravenous dose is required. This term might strictly include the fraction metabolized by cells, like amino acids used by cells as an energy source. The use of stable isotopes is a valid alternative to measure bioavailability and is well suited to study hydrophilic compounds. However, it is technically challenging and expensive. This strategy was used to determine the intact absorption of dipeptides in rats. [START_REF] Nakashima | Application of 13C Stable Isotope Labeling Liquid Chromatography-Multiple Reaction Monitoring-Tandem Mass Spectrometry Method for Determining Intact Absorption of Bioactive Dipeptides in Rats[END_REF] On a rather different tack, to determine protein digestibility, a dual-stable-isotope method was developed, where the ingestion of an intrinsically 2 Hlabeled test protein along with C 13 labeled standard protein of known digestibility permits an accurate measure of digestion and absorption to meet human protein requirements. [START_REF] Devi | Measurement of Protein Digestibility in Humans by a Dual-Tracer Method[END_REF] The body metabolism is a critical factor in measuring nutrient bioavailability. For example, many studies have attempted to measure the bioavailability of bioactive peptides. In a pharmacokinetic study in rats, the transport and distribution of a digestion-resistant casein-derived antihypertensive hexapeptide, HLPLP, showed an absorption rate of 5.2%. The same compound had previously shown a transepithelial flux in the Caco-2 model of less than 1‰, in accordance with the already reported lower absorption values in this cell model. More importantly, the absorption in vivo was followed by metabolization of the peptide into at least three species (HLPL, LPLP, and HLP). This highlights the difficulty in relating bioavailability to bioactivity, as these biotransformed peptides could elicit as well antihypertensive activity. [START_REF] Sánchez-Rivera | Peptide Fragments from β-Casein f(134-138), HLPLP, Generated by the Action of Rat Blood Plasma Peptidases Show Potent Antihypertensive Activity[END_REF] The plasma concentration of similar antihypertensive peptides in humans was on the order of pico-or nanomolar, representing an absorption rate below 0.1%. In another study, MALDI-MS imaging was conducted to visualize the antiatherosclerotic dipeptide WH in the rat intestinal membrane as compared with the reverse sequence HW. Whereas the first sequence was absorbed intact in blood, the dipeptide HW was not, despite similar P app values for both peptides in Caco-2 monolayers. The MS imaging approach allowed the hydrolysis of HW by brush border peptidases at the intestinal membrane. These examples support the need to improve the models to evaluate in vitro permeability. [START_REF] Tanaka | Visualized Absorption of Anti-Atherosclerotic Dipeptide, Trp-His, in Sprague-Dawley Rats by LC-MS and MALDI-MS Imaging Analyses[END_REF] In summary, there is no perfect method available for the determination of the absolute food nutrient bioavailability.

Human studies are costly and invasive and still generate information only on relative bioavailability. In addition, validated methods are required to collect accurate data from these studies. Correlation of in vitro data to in vivo findings is also urgently needed.

MODELS USED IN NUTRITION FOR TESTING INTESTINAL TRANSEPITHELIAL TRANSPORT

The human intestinal barrier consists of different cell types, including enterocytes, goblet, Paneth, tuft, enteroendocrine, and stem cells. They are present in different proportions and densities from proximal to distal intestine, fulfilling not only a physical separation function but also permitting selective sensing and absorption of nutrients, as well as playing a role in intestinal immunological functions. The transport models are intended to mimic the natural epithelial polarity, i.e., presence of an apical or mucosal side and a basolateral or serosal side and capability of the known mechanisms: paracellular, F transcellular, carrier-mediated, or vesicle-mediated (Figure 2). In vivo, this barrier crossing will be governed by the compound structure and concentration of the bioaccessible fraction. A view on the different systems used to mimic the transit of nutrients or contaminants across the intestinal epithelium is presented in order of increasing in vivo representation. Some advantages and limitations of the intestinal models used for transport studies are listed in Table 1.

Artificial Membrane and in Silico

Models. Cell-free artificial barriers respond to the need for a low cost, easy handling, and rapid screening model to predict gastrointestinal transcellular passive permeability. These systems make use of a donor and a receiver/acceptor compartment separated by a barrier of defined pore size, usually a polymeric membrane. The permeability values recorded in drug transport are usually far lower than those from intestinal perfusion experiments; however, they are comparable to experiments where permeability is tested with Caco-2 cell monolayers. [START_REF] O'shea | Best Practices in Current Models Mimicking Drug Permeability in the Gastrointestinal Tract -An UNGAP Review[END_REF] In silico approaches are also often used to predict the permeability mechanistically. Data from PAMPA assays have been subject to numerous quantitative structure-activity relationship (QSAR) studies, and Volsurf computational approaches where the information from 3D interaction energy grid maps is condensed into 2D molecular descriptors for pharmacokinetic modeling. Thus, hydrogen bonding ability in addition to hydrophobicity at a particular pH have been shown to be significant, for example, in determining variations in PAMPA permeability coefficients of natural flavonolignans. [START_REF] Diukendjieva | In Vitro and in Silico Studies of the Membrane Permeability of Natural Flavonoids from Silybum Marianum (L.) Gaertn. and Their Derivatives[END_REF] Another in silico approach was used to rank olefins by a reactivity simulation study. The reactivity was described by the position of the double bond rather than the number of carbon atoms while in the lipophilicity and permeability analysis, both parameter descriptors depended mainly on the number of carbon atoms; these results were predictive of the absorption in an everted sac model. [START_REF] Shi | Assessment of the Intestinal Absorption of Higher Olefins by the Everted Gut Sac Model in Combination with In Silico New Approach Methodologies[END_REF] These computational and membrane models are emerging as alternative methods to animal testing for transcellular passive permeability studies (see for example https://data.jrc.ec.europa.eu/collection/id-0088), but comparison with human absorption needs further attention.

4.2. Cell-Based Models. Various cell-based assays have been adopted to analyze food--intestine interactions and the absorption of dietary compounds. Even though the Caco-2 intestinal barrier model lacks physiological diversity, this cell line represents enterocytes, which are the most numerous cell types in the intestine. Over a period of 21 days, these cells can differentiate into polarized monolayers of absorptive enterocytes with an apical brush border membrane and microvilli. Caco-2 monolayers provide the simplest system to study absorption by paracellular, transcellular, and carrier mediated absorption. In vivo, the gut barrier is protected by a dense mucus layer, which should be penetrated by food molecules as a first stage of absorption. To mimic such a barrier in vitro, a mucus layer can be overlaid on the monolayers or Caco-2 cells can be cocultured with HT29-MTX adding mucus-secreting goblet cells. This coculture showed no toxic effect against biorelevant intestinal fluids in terms of viability and lactate dehydrogenase release, suggesting the apical mucus barrier was cell protective. [START_REF] Antoine | Biorelevant Media Resistant Co-Culture Model Mimicking Permeability of Human Intestine[END_REF] Furthermore, to increase cell representation in the monolayer to track allergens or immunomodulators, immune cells, such as RAW264, PBMCs, Raji B, U937, and THP-1, have been added to Caco-2 or Caco-2/HT29 monolayers. The strong correlation (R 2 = 0.84) between permeability data generated from Caco-2 monolayers and from human trials for drug compounds that utilize transcellular passive transport [START_REF] Jarc | Demonstrating Suitability of the Caco-2 Cell Model for BCS-Based Biowaiver According to the Recent FDA and ICH Harmonised Guidelines[END_REF] makes this model reliable to study this permeability mechanism. However, this cellular model is not expected to provide absolute absorption values, and relevance to in vivo data in relation to other permeability mechanisms must be determined.

Intestinal organoids, also called "mini-guts", are structures grown from stem cells and consist of organ-specific cell types that self-organize and maintain the main physiological characteristics of the intestinal tissue. In this model, a polarized epithelium with tight junctions is developed and includes enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. They are generated from isolated intestinal tissue of mice or porcine immediately after sacrifice or from humans through surgery biopsy and can be maintained in culture in the laboratory over months or preserved under liquid N 2 for longer storage. Although 3D organoids have apicobasal polarity with their apical membranes located toward the organoid core ("basal-out" polarity), apical surfaces are largely inaccessible when stimuli are applied via the media. However, it has been reported that organoids are porous and that solutions can readily reach the apical membrane. Organoids seeded in a 2D layer in bicameral plate increase the relevance of the monolayer and can be cocultured in the presence of gut-derived fibroblasts to add physiological complexity, [START_REF] Schweinlin | Development of an Advanced Primary Human In Vitro Model of the Small Intestine[END_REF] which was applied to evaluate the barrier integrity in the presence of shear stress and dynamic flow conditions, physiological characteristics found in vivo. Other studies evaluated not only the permeability but also the transporter involved in the process. A competitive assay between different cyclic hexapeptides and the radiolabeled Gly-Sar was performed to screen for transporter-mediated uptake of those peptides. [START_REF] Zietek | Organoids to Study Intestinal Nutrient Transport, Drug Uptake and Metabolism -Update to the Human Model and Expansion of Applications[END_REF] The results showed significantly decreased radiolabeled Gly-Sar uptake, with the candidate peptides being substrates for peptide transporter-mediated uptake in humans. This highlights the suitability of intestinal organoids to screen for transporter-mediated uptake, a process under-appreciated in other cellular systems due to poor transporter expression. Because 3D organoids have their apical membranes toward the organoid core, rendering them inaccessible, the use of nonenzymatically dissociated ("broken up") organoids instead of intact ones could be an option to target basolateral and apical transporters at the same time. [START_REF] Zietek | Organoids to Study Intestinal Nutrient Transport, Drug Uptake and Metabolism -Update to the Human Model and Expansion of Applications[END_REF] Similarly, using organoids with an inverse structure (named xenogeneic-free human intestinal organoids), Inoue et al. [START_REF] Inoue | Xenogeneic-Free Human Intestinal Organoids for Assessing Intestinal Nutrient Absorption[END_REF] tested the barrier permeability and showed that the "inside-out" organoids assimilated glucose, dipeptide, and cholesterol through the specific transporters expressed on the cellular surface. Moreover, similar transporter expression (SGLT1, GLUT2, PEPT1, NPC1L1) was found compared to the human intestine. It is important to note that these intestinal tissue-derived organoids have their limitations, as they only consist of epithelial cells and the mesenchyme, the vasculature, neuronal cells, and the immune system are lacking. Also, the epithelial cell composition is dependent on the cocktail of growth factors and cytokines added during culture, which does not necessarily reflect the in vivo epithelial cell composition.

Organ-on-chip (OoC) technology represents a new kind of system useful for understanding the complexity of the human intestine. This technology allows to recapitulated tissue-level physiology and function into biologically based microfluidic in vitro devices, also known as microphysiological systems (MPS). Gut-on-chip systems contain microchannels that are continuously perfused and are covered by different intestinal cell types, arranged to form tissues mimicking the in vivo morphology. They try to mimic not only the mechanical, structural, absorptive, and transport properties, but also pathophysiological ones including for instance intestinal microbes or immune cells. [START_REF] Maurer | A Three-Dimensional Immunocompetent Intestine-on-Chip Model as in Vitro Platform Journal of Agricultural and Food Chemistry pubs[END_REF] Some of the most remarkable advantages of MPS or OoC systems are the peristalsis simulation and flow-induced shear stress. Both biomechanical signals play a fundamental role in cell differentiation. Peristalsis-like strain on cells was reported to induce 3D intestinal villi morphogenesis, [START_REF] Kim | Gut-on-a-Chip Microenvironment Induces Human Intestinal Cells to Undergo Villus Differentiation[END_REF] while shear stress increased mitochondrial activity or mucus production and therefore drug absorption. [START_REF] Delon | A Systematic Investigation of the Effect of the Fluid Shear Stress on Caco-2 cells towards the Optimization of Epithelial Organ-on-Chip Models[END_REF] Several studies show fully matured intestinal cells after 4 days under dynamic flow instead of a 21 day period for static conditions, so these models could be shortened with the advantage that this entails. However, there are technical needs such as the development of more biocompatible materials, the integration of sensors, and the optimization of microfluidic design; hence, their use in nutrient transport is still at its beginning.

4.3. Tissue-Based Models. Ussing chambers (UC) consist of two halves separated by an epithelium (sheet of intestinal mucosa or a monolayer of epithelial cells grown on permeable supports). In the UC, excised intestinal animal tissue segments are placed between the two halves of the chamber and kept under physiological buffer, gas, and temperature conditions. UC thus may allow investigation into the absorption of several bioactive compounds such as dietary peptides that cannot easily be done in vivo. The presence of plasma proteases that further hydrolyze peptides reaching the bloodstream makes the untargeted identification of peptides difficult. By using piglet proximal jejunum extracts mounted in the UC, it was possible to take advantage of the innate proteolytically active brush border present in the tissue and detect the formation of several new peptides in the apical compartment. Only a small percentage of a peptide (from 0.6% to 3.35%) was able to cross the epithelial barrier, [START_REF] Ozorio | The Influence of Peptidases in Intestinal Brush Border Membranes on the Absorption of Oligopeptides from Whey Protein Hydrolysate: An Ex Vivo Study Using an Ussing Chamber[END_REF] most of them relating to hydrophobic, aromatic, and acidic residues. The results challenge the common viewpoint relating to the preferred absorption of amino acids, dipeptides, and tripeptides and support the increasing evidence of intact peptide absorption in humans. The permeability of dietary carotenoids was compared between those of mouse intestinal tissue and Caco-2 monolayers. The use of murine mucosal tissue demonstrated a nearly 2-fold uptake for all-trans-β-carotene from orange-fleshed sweet potato as compared to the Caco-2 cells. This murine data compares well with published human in vivo data placing the tissue model as a more realistic approach. [START_REF] Kalungwana | An Ex Vivo Intestinal Absorption Model Is More Effective than an in Vitro Cell Model to Characterise Absorption of Dietary Carotenoids Following Simulated Gastrointestinal Digestion[END_REF] Another system often used ex vivo is the everted small intestinal sac method, which consists of a small piece of intestine removed from anesthetized rats, flushed with buffer, and everted over a tube with the serosal part inside the sac and the mucosal side facing the buffer solution. Both ends are tied and filled with oxygenated buffer and accumulation of the sample in the inner compartment is measured. In a study which compared the transport of egg white digests, a higher permeability was measured in the everted rat sacs than in the Caco-2 cell monolayer, with lower brush border enzyme degradation occurring in the cell model. [START_REF] Wang | Hydrolysis and Transport of Egg White-Derived Peptides in Caco-2 Cell Monolayers and Everted Rat Sacs[END_REF] However, there were some similarities; namely, four peptides with a molecular size of 8-9 amino acid residues were identified in the transported fraction in both models. The fact that both models permitted the transport of these rather long peptides increases the likelihood that these peptides are absorbed in vivo, although no in vivo data is currently available. Noneverted version of small intestinal sac is used as well, with permeability of passively transported molecules similar to that in everted sacs and permeability of actively transported molecules being lower. [START_REF] Ruan | Prediction of Human Absorption of Natural Compounds by the Non-Everted Rat Intestinal Sac Model[END_REF] Apparent permeability of 11 marketed drugs and 13 bioactive natural compounds in noneverted small intestinal sacs demonstrated strong correlation with reported in the literature data on human fraction absorbed. Overall, both of these methods increase throughput in absorption studies, require fewer animals, and have a lower amount of studied compound compared to the animal studies, being more feasible at early stages of drug discovery or food product development.

THE NEED TO STANDARDIZE IN VITRO MODELS

The absorption models based on Caco-2 alone or in combination with other intestinal cells are the most popular option in nutrition. However, the comparability of the assays between laboratories remains unknown due to the large variety of food compounds and conditions assayed. It is generally recognized that the cell source and the culture procedure can have an impact on the permeability as well as on the monolayer integrity. Thus, the heterogeneity in transporter activity of several Caco-2 clones was ascribed to differences in transporter expression, as shown for PepT1 and MDR1, and this was determined by the culture conditions. [START_REF] Hayeshi | Comparison of Drug Transporter Gene Expression and Functionality in Caco-2 Cells from 10 Different Laboratories[END_REF] Moreover the biocompatibility of complex samples such as food digesta with intestinal cell models needs particular attention. [START_REF] Kondrashina | Coupling in Vitro Food Digestion with in Vitro Epithelial Absorption; Recommendations for Biocompatibility[END_REF] This has led this group of researchers to work on the standardization of an in vitro model to be applied to such substrates. First, it is necessary to have a shared procedure for the manipulation of the intestinal cell lines as well as for the preparation of the cell monolayers ready for absorption experiments. For example, the source of culture media and supplements, specifically fetal bovine serum, are critical, as are the type of plastic material used and the method(s) used to determine the permeability of the cellular monolayer, among others. The source of the cell lines and their maintenance can also introduce important variability. To check this, seven groups working within the INFOGEST network made their cell lines available to determine the permeability of the generated monolayers by both automated impedance-based and manual TEER measurement (experimental details in Supporting Information "INFOGEST inter-lab TEER determination of Caco-2 and HT29-MTX monolayers") at IPLA-CSIC, Spain. A total of 11 clones, seven Caco-2 clones and four HT29-MTX, were analyzed. The results showed a strong effect of cell clone on TEER values, regardless of the equipment used for the measurement of this parameter (Figure 3). Values between Caco-2 clones could differ up to 50 times with the automated impedance measurement or 15 times with the manual ohmmeter. The differences in HT29-MTX were lower. Therefore, the origin and subsequent management of the Caco-2 and HT29-MTX clones are a source of variation and an important consideration in the proposal of a standardized absorption procedure. It is therefore critical to suggest a basal range of values that will indicate and validate the permeability state of the monolayer before undertaking any absorption experiment.

Nutrient/contaminant bioavailability is a complex multiorgan process involving several cell types and regulatory mechanisms. Several models of different physiological relevance are used to screen and study intestinal absorption, where the impact of the nature of the food compound is crucial. Current cell-based models are likely to generate results that may not be fully predictive of true bioavailability in humans. Thus, they generally do not account for the strong intersegmental variability, the variability within the population (e.g., children, elderly people, pharmacological therapies, pathologies, sex, hormonal fluctuations, etc.), as well as the interindividual variability. Animal models can also be problematic, as metabolic conversion and absorption processes are often different. However, in vitro models are a good starting point to provide a basis for mechanistic data on absorption and can minimize the use of animal experiments. A joint effort to agree and standardize robust protocols of intestinal transport studies of food compounds is of paramount importance to value the significance of human intestinal cell-based assays and allow the comparison of results from different laboratories. However, values of absorption in vivo will always be necessary to further improve in vitro methods.

■ ASSOCIATED CONTENT

* sı Supporting Information

Figure 1 .

 1 Figure 1. Outline of the specific transport mechanisms described for macronutrients through the intestinal epithelium.

Figure 2 .

 2 Figure 2. Graphical overview of the main models used for transport studies. Apical compartment: intestinal lumen, mucosa, donor; basolateral compartment: systemic circulation, serosa, acceptor). Green arrow indicates the absorption/secretion direction.

Figure 3 .

 3 Figure 3. TEER values of the Caco-2 and HT20-MTX clones from different laboratories analyzed after 21 days of differentiation at IPLA-CSIC by automated impedance-based (CellZscope+) and manual ohmmeter measurement (Millicell ERS).

Table 1 .

 1 Advantages and Disadvantages of Different Cell-Based and Tissue-Based Intestinal Models, As Well As Several Common Alternative Intestinal Models Used for Transport Studies a

a iPSC, inducible pluripotent stem cell; ASC, adult stem cell; N/A, not applicable.

I Químicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal Lorena Ruiz -Dairy Research Institute of Asturias (IPLA-CSIC), Villaviciosa 33300 Asturias, Spain Marta Santos-Hernández -Wellcome Trust -MRC Institute of Metabolic Science, Metabolic Research laboratories, Addenbrooke's Hospital, CB2 0QQ Cambridge, U.K. Laura Soriano-Romaní -AINIA in Vitro Preclinical Studies Area, Parque Tecnológico de Valencia, E46980 Paterna, Spain Lidia Tomás-Cobos -AINIA in Vitro Preclinical Studies Area, Parque Tecnológico de Valencia, E46980 Paterna, Spain Santiaga María Vivanco-Maroto -Institute of Food Science Research CIAL (CSIC-UAM), 28049 Madrid, Spain Isidra Recio -Institute of Food Science Research CIAL (CSIC-UAM), 28049 Madrid, Spain; orcid.org/0000-0003-2973-5329 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jafc.3c05479

Notes

The authors declare no competing financial interest.


domain: info phys stat

text:
Ali Yassin

Hocine Cherifi

Hamida Seba

Olivier Togni

Olivier Togni An

An Empirical Investigation of Statistical Backbone Filtering Techniques

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

An Empirical Investigation of Statistical Backbone Filtering Techniques

Ali Yassin Hocine Cherifi Hamida Seba Olivier Togni University of Burgundy, FRANCE University of Burgundy, FRANCE University of Lyon, FRANCE University of Burgundy, FRANCE ali_yassin@etu.u-bourgogne.fr hocine.cherifi@u-bourgogne.fr hamida.seba@univ-lyon1.fr olivier.togni@u-bourgogne.fr • Marginal Likelihood Filter each unit edge randomly chooses two nodes, which results in a binomial distribution.

• ECM Filter

Employs a null model based on the canonical maximumentropy ensemble of weighted networks having the same degree and strength distribution as the original network.

• LANS Filter Employs the empirical cumulative density function to judge statistical significance.

• Disparity Filter a node distributes its strength (summation of weights)

uniformly on the edges.

• Polya Urn Filter a node distributes its strength at random through a Polya process, which results in a Beta-binomial distribution.

• GloSS Filter edge weights are randomly drawn from the empirical weight distribution.

What is a Network Backbone? What about the distributions?

This work is funded by Agence Nationale de Recherche under grant ANR-20-CE23-0002

	Filter	Technique Behavior	Giant Component	Isolated Nodes	Weights Retained
	Marginal Likelihood	Conservative	Always	Low	High
	Noise Corrected	Conservative	Always	Low	Small & High
	ECM	Flexible	α > 10 -2	α < 10 -2	Small & High
	LANS	Aggressive	Always	Low	High
	Disparity	Aggressive	α > 10 -2	High	High
	Polya Urn	Aggressive	α > 10 -2	High	Small & High
	GloSS	Aggressive	α > 10 -2	High	Small & High