Jerome M Capannelli

Sara Dalle Vacche

Alessandra Vitale

Khaoula Bouzidi

Davide Beneventi
email: davide.beneventi@pagora.grenoble-inp.fr

Roberta Bongiovanni
email: roberta.bongiovanni@polito.it

A biobased epoxy vitrimer/cellulose composite for 3D printing

Keywords: Vitrimer, biobased composites, addi ve manufacturing, cellulose, liquid deposi on modeling

Conven onal epoxy polymers are obtained from oil-based resources and their structure is formed by permanent covalent crosslinks. Therefore, this class of materials is now considered non environmentally friendly as they are neither renewable nor reprocessable and recyclable. In this study, we prepared a vitrimeric material based on an epoxy-func onalised cardanol cured with a biobased polycarboxylic acid. In the presence of a zinc-containing catalyst, a so polyester with a Tg=-13 °C was obtained; its reprocessability by a chemical method was demonstrated to be feasible without any relevant change in proper es once a second curing cycle was completed. The vitrimeric polyester was then used in combina on with cellulose powder for the prepara on of a sustainable and biobased composite. The matrix/filler mass frac on was tailored to obtain a composite paste with suitable rheological proper es for 3D prin ng via Liquid Deposi on Modelling (LDM). Preliminary prin ng tests were successful, and the vitrimeric printed parts were then thermally cured retaining the shape. The suitability of the vitrimeric composite for addi ve manufacturing was thus confirmed: the new material can provide a solu on to 3D prin ng of recyclable thermose ng biobased polymers.

Introduction

The demand for more sustainable polymeric materials, whether biobased and/or recyclable, has been increasing for decades. In this scope, subs tu on of thermosets, which are permanently crosslinked polymers and cannot be reprocessed nor reshaped, is required. To this aim, new types of polymeric networks called Covalent Adaptable Networks (CANs) or Dynamic Covalent Networks (DCNs) have been developed (N. Zheng et al. 2021;[START_REF] Kloxin | Covalent Adaptable Networks (CANs): A Unique Paradigm in Cross-Linked Polymers[END_REF]; Z. P. [START_REF] Zhang | Polymer Engineering Based on Reversible Covalent Chemistry: A Promising Innova ve Pathway towards New Materials and New Func onali es[END_REF][START_REF] Winne | Dynamic Covalent Chemistry in Polymer Networks: A Mechanis c Perspec ve[END_REF]: CANs can be defined as polymeric materials that exhibit covalent crosslinks that are reversible under a specific s mulus, such as heat (J. Zheng et al. 2021), thus retaining the performances of thermosets and combining them with the ability of thermoplas cs of being malleable and reprocessable. In detail, CANs are classified into two groups, associa ve and dissocia ve CANs [START_REF] Podgórski | Toward S muli-Responsive Dynamic Thermosets through Con nuous Development and Improvements in Covalent Adaptable Networks (CANs)[END_REF]. For dissocia ve CANs, recyclability is assured by dissocia ve exchange mechanisms, such as Diels-Alder reac ons: dissocia on causing a drop in crosslinking density brings fluidity and thus reprocessability, but it is detrimental to material performance, e.g., solvent resistance is usually lost. On the contrary, associa ve CANs are based on dynamic exchange mechanisms without any change in crosslinking density: different bonds are broken and formed at the same me, which allows maintaining the material performance unchanged, e.g., solvent resistance. The breakthrough regarding the class of associa ve CANs was the synthesis of vitrimers by Leibler's group in 2011 [START_REF] Montarnal | Silica-Like Malleable Materials from Permanent Organic Networks[END_REF]). The word "vitrimer" was suggested by observing that the polymer showed a viscosity change upon hea ng as vitreous silica materials. Then a characteris c temperature was defined, called topology freezing transi on temperature Tv, above which the material flows and its viscosity changes following an Arrhenius trend. For the development of vitrimers, many exchangeable reac ons have been inves gated, such as disulfide [START_REF] Guggari | Vanillin-Based Epoxy Vitrimers: Looking at the Cystamine Hardener from a Different Perspec ve[END_REF]Azcune et Odriozola 2016;[START_REF] De Luzuriaga | Epoxy Resin with Exchangeable Disulfide Crosslinks to Obtain Reprocessable, Repairable and Recyclable Fiber-Reinforced Thermoset Composites[END_REF][START_REF] Chen | Rapid Stress Relaxa on and Moderate Temperature of Malleability Enabled by the Synergy of Disulfide Metathesis and Carboxylate Transesterifica on in Epoxy Vitrimers[END_REF], imine bonds [START_REF] Dhers | A Fully Bio-Based Polyimine Vitrimer Derived from Fructose[END_REF]Taynton et al. 2014;[START_REF] Schoustra | Molecular Control over Vitrimerlike Mechanics -Tuneable Dynamic Mo fs Based on the Hamme Equa on in Polyimine Materials[END_REF], vinylogous urethane [START_REF] Denissen | Vinylogous Urethane Vitrimers[END_REF], boronate ester (Cromwell, Chung, et Guan 2015;[START_REF] Ogden | Recyclable, Strong, and Highly Malleable Thermosets Based on Boroxine Networks[END_REF] or hindered urea [START_REF] Denissen | Vinylogous Urea Vitrimers and Their Applica on in Fiber Reinforced Composites[END_REF][START_REF] Zhang | S muli-Mild, Robust, Commercializable Polyurethane-Urea Vitrimer Elastomer via N,N -Diaryl Urea Crosslinking[END_REF], in addi on to transesterifica on reac ons [START_REF] Capelot | Cataly c Control of the Vitrimer Glass Transi on[END_REF][START_REF] Fang | Vitrimer Chemistry Enables Epoxy Nanocomposites with Mechanical Robustness and Integrated Conduc ve Segregated Structure for High Performance Electromagne c Interference Shielding[END_REF][START_REF] Shi | Recyclable 3D Prin ng of Vitrimer Epoxy[END_REF]), which are s ll the most exploited since the first work on epoxy vitrimers [START_REF] Montarnal | Silica-Like Malleable Materials from Permanent Organic Networks[END_REF]. In these systems, the reac on can involve either the ester groups formed during the hardening reac on of acids or anhydrides and epoxides or those ini ally present in the epoxy/hardener backbones. The polymer recycling based on transesterifica on can be a mechanical or a chemical process. In the first case, the vitrimer is grinded and then reshaped with a compression moulding machine at high temperature and high pressure, such as 180 °C and 40 bar [START_REF] Fang | Vitrimer Chemistry Enables Epoxy Nanocomposites with Mechanical Robustness and Integrated Conduc ve Segregated Structure for High Performance Electromagne c Interference Shielding[END_REF]). This approach is the most straigh orward, but costly; moreover, if a composite must be treated, it implies damage to the reinforcement phase. Alterna vely, the vitrimer can be chemically recycled by dissolu on by an alcohol such as ethylene glycol, or water: the solvent swells the network and takes part in the transesterifica on reac on cleaving the chains. A er dissolu on, by hea ng the system at high temperature (around 180 °C), the solvent evaporates and the crosslinked structure is restored [START_REF] Shi | Solvent Assisted Pressure-Free Surface Welding and Reprocessing of Malleable Epoxy Polymers[END_REF][START_REF] Fang | Vitrimer Chemistry Enables Epoxy Nanocomposites with Mechanical Robustness and Integrated Conduc ve Segregated Structure for High Performance Electromagne c Interference Shielding[END_REF]. Therefore, epoxy vitrimers are not only reprocessable as thermoset, but they can be processed and reprocessed as thermoplas cs through conven onal shaping processes, i.e., extrusion and injec on moulding. As the development of epoxy vitrimers targets materials having large industrial interest and commercial relevance, their implementa on is likely to be simple and with a high impact making a great change in industry. Moreover, in the case of composites, the thermally ac vated bond exchange via transesterifica on, which allows the polymeric matrix to flow at high temperature, gives the op on of recycling the material as it is or isola ng the matrix and reclaiming the filler [START_REF] Fang | Vitrimer Chemistry Enables Epoxy Nanocomposites with Mechanical Robustness and Integrated Conduc ve Segregated Structure for High Performance Electromagne c Interference Shielding[END_REF][START_REF] Kuang | Recycling of Epoxy Thermoset and Composites via Good Solvent Assisted and Small Molecules Par cipated Exchange Reac ons[END_REF]. Further interest for these innova ve epoxy networks is their applica on in the field of addi ve manufacturing (AM) (Perzhada et al. 2020). At present, common AM technologies for epoxides are stereolithography and related techniques, and ink wri ng, employing photosensi ve formula ons as common prac ce. Thermoset epoxides are also demonstrated suitable for je ng techniques, and few examples concerning extrusion techniques are found in literature, even applied to epoxy-based vitrimers using adapted Fused Deposi on Modelling [START_REF] Shi | Recyclable 3D Prin ng of Vitrimer Epoxy[END_REF][START_REF] Peerzada | Addi ve Manufacturing of Epoxy Resins: Materials, Methods, and Latest Trends[END_REF]. However, to the best of our knowledge, no examples of processing epoxy vitrimers by cold Liquid Deposi on Modelling (LDM), are present in the literature. LDM is an extrusion-based 3D prin ng technique [START_REF] Valino | Advances in 3D Prin ng of Thermoplas c Polymer Composites and Nanocomposites[END_REF] and is currently applied for the processing of ceramics, wood and food pastes. However, it is a rac ve compared to the other AM technologies cited above, as it has lower costs, uses a simple set up working at room temperature, and has good poten al for producing large size pieces. Besides developing applica ons by 3D prin ng, a further challenge in the field of vitrimers is the use of sustainable raw materials. Different groups reported the synthesis of par ally or fully biobased vitrimers [START_REF] Guggari | Vanillin-Based Epoxy Vitrimers: Looking at the Cystamine Hardener from a Different Perspec ve[END_REF]) star ng from vegetable oils, lignin deriva ve such as vanillin [START_REF] Guggari | Vanillin-Based Epoxy Vitrimers: Looking at the Cystamine Hardener from a Different Perspec ve[END_REF] or furan-based precursors [START_REF] Dhers | A Fully Bio-Based Polyimine Vitrimer Derived from Fructose[END_REF]. Few interes ng reviews were recently published on the topic [START_REF] Zhao | Biobased Covalent Adaptable Networks: Towards Be er Sustainability of Thermosets[END_REF][START_REF] Vidil | Fully Biobased Vitrimers: Future Direc on toward Sustainable Cross-Linked Polymers[END_REF][START_REF] Lucherelli | Biobased Vitrimers: Towards Sustainable and Adaptable Performing Polymer Materials[END_REF]. In the case of epoxy vitrimers based on transesterifica on reac ons, they can be synthesized using biobased epoxy resins and biobased hardeners, where the ester groups are either present in the backbone of the precursors or are formed during their hardening, i.e., employing acids or anhydrides as co-reactants [START_REF] Zhao | Biobased Covalent Adaptable Networks: Towards Be er Sustainability of Thermosets[END_REF][START_REF] Zhang | Recyclable, Repairable and Malleable Bio-Based Epoxy Vitrimers: Overview and Future Prospects[END_REF][START_REF] Chong | A Review on Recent Approaches to Sustainable Bio-Based Epoxy Vitrimer from Epoxidized Vegetable Oils[END_REF]. Considering the perspec ve of applying epoxides in AM and taking into account the demand of sustainability, in this work we prepared an environmentally benign epoxy vitrimer composite suitable for extrusion 3D prin ng: to this aim we employed precursors with a high biobased content and cellulosic powder both as reinforcement and rheology modifier. First, the biobased epoxy vitrimer matrix was synthesized, and its chemical recycling was inves gated; then, by adding cellulose powder, the rheological behaviour of the composite was tuned and printability by LDM process was assessed fabrica ng a simple demonstrator.

Materials and Methods

Materials

The synthesized epoxy vitrimer was based on epoxidized cardanol NC-514S, supplied by Cardolite®, with an epoxide equivalent weight of 438 g/eq and average func onality f= 1.1 [START_REF] Jaillet | New Biobased Epoxy Materials from Cardanol: New Biobased Epoxy Materials from Cardanol[END_REF]. A mixture of fa y dicarboxylic and tricarboxylic acid, Pripol 1040, with an acid equivalent weight of 296 g/eq and 100% biobased content, was used as crosslinker and kindly provided by Croda Ltd. The chemical structure of the epoxy precursor and of the dimer/trimer acid is reported in Figure 1. The catalyst used was zinc acetate Zn(Ac)2 and was purchased from Carl Roth. For the composite, a commercial cellulose powder, Technocel® FM8, was supplied by CFF GmbH & Co. KG and used as a filler without any pretreatment. According to the datasheet, par cle diameter is reported to be between 6 and 12 µm, as also confirmed in a previous work [START_REF] Bouzidi | 3D Printable Fully Biomass-Based Composite Using Poly(Furfuryl Alcohol) as Binder and Cellulose as a Filler[END_REF]. Acetone (>= 99.0% pure) was supplied by Acros Organics, ethylene glycol was purchased from Carl Roth. All the products were used as received.

Preparation and curing of the vitrimer

The vitrimer was prepared in a single step: the epoxidized cardanol NC-514S, the dimer/trimer acid and the catalyst Zn(Ac)2, either with or without cellulose powder, were mixed in a planetary mixer (KitchenAid 5KSM3311X) at room temperature at 120 rpm for 5 min and then at 200 rpm for addi onal 5 min. Before any curing, the samples were stored in the fridge at 4-5 °C to prevent any crosslinking during storage. The systems were thermally cured in oven at 200 °C for 6 h a er pouring the mixture in a PTFE mould. In detail, NC-514S and acid were mixed maintaining an equimolar ra o epoxide/COOH groups. Zn(Ac)2 concentra on was 10 mol% with respect to acid groups. In a typical formula on of 100 g, 58.21 g of NC-514S, 39.35 g of Pripol 1040 and 2.44 g of Zn(Ac)2 were mixed. Cellulose powder weight frac on was in the range of 30-42wt% with respect to the total mass of the composite.

3D Printing

The 3D prin ng was performed by LDM using a commercial benchtop 3D printer (Ar llery Sidewinder X1) a er proper customiza on. The printer, originally designed to print fused filaments, was upgraded by adding a screw extruder (WASP claystruder) with a nozzle diameter of 0.7 mm, fed by a syringe under pressure at 3 bars to ensure proper feeding. The so ware Simplify3D® was used as slicer. A er the flow calibra on of the screw extruder, 3D prin ng was carried out with a fixed layer height of 0.4 mm, a prin ng speed of 500 mm/min and infill of 60 % with 45/-45 ° angles. A er prin ng, the objects were thermally cured following the same thermal process applied for the neat vitrimer, i.e., hea ng at 200 °C for 6 h.

Vitrimer chemical recycling

The recycling process was performed adap ng the procedure described in [START_REF] Shi | Recyclable 3D Prin ng of Vitrimer Epoxy[END_REF]. The cured samples were cut in pieces of around 10 x 10 x 5 mm 3 and immersed in a bath of ethylene glycol that was heated at 180 °C under reflux for 6 h. A er dissolu on, the obtained solu on was dis lled for 6 h at 180 °C under vacuum to recycle both the vitrimer and the ethylene glycol.

Characterization

Fourier Transform Infrared (FT-IR) analysis was performed in a enuated total reflec on (ATR) mode (PerkinElmer, Spectrum65) using the universal ATR sampling accessory equipped with a diamond crystal. The spectra were acquired in the 600-4000 cm -1 range, with 32 scans per spectrum and a resolu on of 4 cm -1 . Thermogravimetric analysis (TGA) was performed using a TGA/DSC 3+ StarSystem from Me ler Toledo. Measurements were performed from 25 °C to 800 °C with a hea ng rate of 10 °C/min, under N2 flux, preven ng any thermo-oxida ve process, and from 800 °C to 900 °C with a hea ng rate of 10 °C/min, under air flux. First order deriva ve of the weight profile was calculated by Origin (OriginLab) so ware to be er observe the main degrada on temperatures of the produced samples. Differen al scanning calorimetry (DSC) was performed using a DSC Q100 from TA Instrument. The measurements were conducted making a first hea ng at 20 °C/min from 25°C to 100 °C to remove any thermal history in the sample, followed by two cycles from -100 °C to 280 °C at a hea ng rate of 10 °C/min under N2 flux. Glass transi on temperature was taken as the inflec on point in the second cycle. Insoluble frac on and solubility tests were performed in acetone. Samples of around 0.5-1 g were wrapped in a metallic net with a mesh size of 100 µm and soaked in acetone for 24 h, then they were le drying for 48 h and the mass loss of the samples was calculated. Rheological analyses were performed using an Anton Paar rota onal rheometer MCR302 equipped with parallel plates (= 25 mm, gap = 1 mm). Apparent viscosity was measured in the shear rate range of 0.1 s -1 to 100 s -1 . Viscoelas c proper es were measured on the same instrument with the Amplitude Sweep Test at a frequency of 1 Hz and strain ranging from 0.001 % to 100 %. The evolu on of both the storage modulus (G') and the loss modulus (G'') as a func on of stress was analysed.

Relaxa on tests were performed on the same rota onal rheometer using cured samples with  = 40 mm and 1 mm thick gap. The sample was placed between parallel plates and contact was ensured by applying a 10 N normal force. A er the set temperature was reached, the sample was equilibrated for 20 minutes before star ng the measurement. Relaxa on measurements were performed by applying a shear strain of 2 % in the linear viscoelas c region of the material.

Results and Discussions Preparation and characterization of biobased vitrimer

The biobased epoxy vitrimer was synthesized using a commercial biobased resin (NC-514S), obtained by epoxida on of cardanol, a phenolic lipid found in the cashew nutshell liquid (CNSL), and a mixture of dicarboxylic and tricarboxylic fa y acids having 100 % renewable carbon content (Pripol 1040) as a hardener. Their chemical structures are shown in Figure 1. The curing of the resin, leading to the biobased vitrimer, was made by hea ng at 200 °C for 6 h, in the presence of Zn(Ac)2. At the end of the treatment, a solid was obtained with a highly insoluble content (90.2 %, in acetone), while the star ng raw materials were fully soluble in acetone. As described by Demongeot [START_REF] Demongeot | Coordina on and Catalysis of Zn 2+ in Epoxy-Based Vitrimers[END_REF]) and sketched in Figure 2, the crosslinking process is due to the reac on of the oxirane rings of the mul func onal epoxide NC-514S with the carboxylic groups of the hardener Pripol 1040, forming ester bonds. The comple on of the esterifica on reac on was controlled by ATR-FT-IR spectroscopy. Figure 3 reports the spectra of the epoxy/acid system before and a er hea ng (i.e., curing): we can easily observe the complete disappearance of the band related to epoxide at 910-915 cm -1 , the shi of the band a ributed to the carboxylic acids from 1715 cm -1 to a higher value, around 1740 cm -1 , related to ester groups, and addi onally the appearance of a broad peak at around 3400 cm -1 due to hydroxyl groups. All these results assess the ring opening of the oxirane groups and their esterifica on, clearly confirming the successful crosslinking of the epoxy resin. The obtained cured solid polyester (i.e., vitrimer) was characterized by thermal analyses, i.e., DSC and TGA. In Figure 4, the DSC thermograms of the material before and a er curing are compared. The uncured epoxy/acid system shows a glass transi on temperature (Tg) at around -50 °C, which is comparable to the Tg of pure epoxy resin, known to be around -48 °C (Dalle Vacche, Vitale, et Bongiovanni 2019). A er the hea ng cycle, the crosslinked polyester has a higher Tg at -13 °C, as expected: the network is however in its rubbery state at room temperature and at the common service temperatures. In the DSC curve of the uncured polymer (Figure 4) above 150 °C, an exothermic broad peak is present, which disappears upon hea ng: this signal is related to the enthalpy of the esterifica on reac on and its disappearance confirms the comple on of the crosslinking. The heat of the esterifica on reac on obtained by the signal integra on was used to evaluate the enthalpy value of the epoxy ring opening reac on of the system under inves ga on: the H value is 56 kJ/mol, which is rather close to the mean value of the molar reac on enthalpy reported in the literature for commercial epoxides, i.e., 65 ± 3 kJ/mol [START_REF] Kretzschmar | Reac on Enthalpies During the Curing Of Epoxy Resins with Anhydrides[END_REF]. Figure 5 shows the TGA curve of the cured vitrimer and of the pure epoxy resin (NC-514S). As reported in Table 1, upon hea ng under inert atmosphere, the cured vitrimer shows the onset of the degrada on (T5, i.e., the temperature corresponding to a mass loss of 5 %) at 319 °C, while the main degrada on happens at a higher temperature, and T50 (i.e., the temperature corresponding to a mass loss of 50 %) is at 443 °C. Compared to the pure liquid resin NC-514S, the degrada on of the crosslinked polymer is shi ed to higher temperature as expected. Addi onally, data reported in Table 1 show that the cured polyester has a higher residual mass at 800 °C, ca. 6 %, than the pure resin, further sugges ng that the crosslinking between the epoxy and the acid took place. Hea ng under air up to 900 °C the organic ma er is thermally oxidized, for the pure resin the residue is zeroed, while the value for the crosslinked polymer decreases to ca. 3 %, slightly higher than the ini al mass of catalyst. It was demonstrated in many works [START_REF] Kloxin | Covalent Adaptable Networks (CANs): A Unique Paradigm in Cross-Linked Polymers[END_REF]) that covalent adaptable networks show temperature-frequency-dependency of the storage (G ) and loss (G ) moduli, while thermosets formed by permanent covalent bonds display a temperature-frequency-independent storage (G ) modulus. Therefore, we studied the viscoelas city of our network by stress relaxa on experiments at different temperatures, applying a small deforma on (2 %) and adop ng the Maxwell model to es mate the relaxa on me τ (Eq. 1):

= 𝑒 Eq. 1
where G0 is the ini al value of the storage modulus, t is the me, and the relaxa on me τ corresponds to the me for which G'/G0 = 1/e = 0.37.

In Figure 6A, the G' values for the cured vitrimer sample at different temperatures, normalized over G0, are reported as a func on of me: the elas c response of the cured polyester is clearly changing depending on me and temperature, thus exhibi ng the behaviour of an adaptable covalent network.

As expected, we observed faster stress relaxa on at higher temperatures (Figure 6A): the relaxa on me of our vitrimer ranges from ≈ 1200 s at 200 °C up to ≈ 10000 s at 160 °C. These relaxa on mes are longer than those for epoxy vitrimers based on the most common epoxide, bisphenol-A diglycidyl ether (DGEBA), cured with the same fa y acids in the same equimolar ra o as in the present work: for the DGEBA based network τ ≈ 300 s at 200 °C and τ ≈ 1500 s at 160 °C [START_REF] Shi | Solvent Assisted Pressure-Free Surface Welding and Reprocessing of Malleable Epoxy Polymers[END_REF]. This is a striking difference: as the molecular mechanism of the underlying chemical exchange was the same, having used the same catalyst at a slightly higher concentra on, one could even expect a faster reac on than for the DGEBA vitrimer. It is evident that other parameters can possibly govern the dynamic proper es of epoxy vitrimers and in recent papers they were iden fied as chain mobility, synergies of different dynamic bonds and density of exchangeable bonds [START_REF] Yu | Influence of stoichiometry on the glass transi on and bond exchange reac ons in epoxy thermoset polymers[END_REF][START_REF] Taketo | Cri cal Effects of Branch Numbers at the Cross-Link Point on the Relaxa on Behaviors of Transesterifica on Vitrimers[END_REF][START_REF] Hayashi | Fair Inves ga on of Cross-Link Density Effects on the Bond-Exchange Proper es for Trans-Esterifica on-Based Vitrimers with Iden cal Concentra ons of Reac ve Groups[END_REF]]. In our case, looking at the structures of the polyesters under comparison, presence of different dynamic bonds and neighbouring group par cipa ng and/or influencing the exchange reac on of DGEDA polyester cannot be found. Meanwhile chain mobili es are different: as the aroma c DGEBA network has a higher Tg (i.e., 33°C versus -13°C) than the novel biobased polyester, the la er should show an easier molecular diffusion, favouring transesterifica on, which is not. The density of the exchangeable bonds is another difference between the networks: the inves gated biobased vitrimer has a much lower crosslinking density e than DGEBA [START_REF] Shi | Solvent Assisted Pressure-Free Surface Welding and Reprocessing of Malleable Epoxy Polymers[END_REF]) due to the difference in func onality (Cardolite has an average func onality f=1.1, with epoxy equivalent weight =438 g/eq, while DGEBA has f=2, with epoxy equivalent weight =172 g/eq) being the epoxy-acid conversion quan ta ve for both systems (the calcula on of e for the inves gated vitrimer network can be found in the Suppor ng Informa on). The effect of the e value on relaxa on me is discussed in detail for model systems of epoxides cured with Pripol by Chen [START_REF] Chen | The Crucial Role in Controlling the Dynamic Proper es of Polyester-Based Epoxy Vitrimers: The Density of Exchangeable Ester Bonds[END_REF] and it is demonstrated that the typical stress relaxa on behaviour of epoxy vitrimers is accelerated increasing crosslinking density e. Thus, the lower concentra on of ester bonds of the fully biobased vitrimer prepared from Cardolite epoxide and a biobased acid causes longer relaxa on mes with respect to the DGEBA polyester formed with the same acid. Overall, the choice of a proper f is likely to be crucial for designing a new vitrimer As in vitrimers bonds are only broken if new ones are already formed as a single thermally ac vated process, the relaxa on me of the material changes with the temperature according to the Arrhenius law [START_REF] Sperling | Introduc on to Physical Polymer Science[END_REF], as in Eq. 2:

𝜏 = 𝑒 Eq. 2
where Ea is the ac va on energy of the exchange reac on, T is the temperature, R is the universal gas constant, and k is the pre-exponen al factor. The relaxa on me at different temperature as obtained from the G'/G0 curves are plo ed in Figure 6B: the slope of the fi ng curve corresponds to the coefficient Ea/R and thus Ea (kJ mol -1 ) of the thermal process can be es mated. The Ea value has prac cal importance, as the higher it is the more rapid the decrease of viscosity and the faster the stress relaxa on with rising temperatures: this means dimensional stability at the service temperature of the material and a faster processing on demand. In our case the ac va on energy for the bonds exchange is found Ea ≈ 85 kJ mol -1 and is in agreement with the literature data for epoxy networks (J. Zheng et al. 2021;[START_REF] Altuna | Epoxy Vitrimers with a Covalently Bonded Ter ary Amine as Catalyst of the Transesterifica on Reac on[END_REF][START_REF] Montarnal | Silica-Like Malleable Materials from Permanent Organic Networks[END_REF][START_REF] Chen | The Crucial Role in Controlling the Dynamic Proper es of Polyester-Based Epoxy Vitrimers: The Density of Exchangeable Ester Bonds[END_REF]. This means that the material under inves ga on can compete with tradi onal epoxy vitrimers. From the viscoelas city studies, the topology freezing temperature, Tv, of the polymer, was also es mated: it is defined as the temperature of transi on of a material from a viscoelas c solid to a viscoelas c liquid. As this transi on is considered to happen for a viscosity η of 10 12 Pa s, first the corresponding relaxa on me τ* can be calculated from the Maxwell model by Eq. 3:

𝜂 = 𝐺𝜏* Eq. 3
obtaining G from the value of the storage modulus E' at the rubbery plateau according to Eq. 4:

𝐺 = ( )
Eq. 4

where ν is the Poisson's ra o of the material, which can be assumed =0.5 for rubbery materials. The value of E' was obtained by dynamic mechanical analyses (DMA), which are reported in Figure S1 of the Suppor ng Informa on. Once the relaxa on me τ* was es mated by using Eq. 3, through the rela onship ln(𝜏) versus 1000/T (see Figure 6B), the value of Tv for the vitrimer network was found 71°C (see the Suppor ng Informa on for the calcula ons).

Vitrimer chemical recycling

As already men oned previously, the main interes ng feature of CANs is their ability to be recycled/reprocessed. In this work, we performed the chemical recycling of the biobased epoxy vitrimer leveraging the transesterifica on reac on with ethylene glycol (EG). As depicted in Figure 7, a er dissolu on of the cured material in EG, the solvent was par ally evaporated at high temperature to obtain a resin with the desired viscosity. Then, by further hea ng, the remaining solvent was dis lled, and the crosslinked network was rebuilt with the aid of the Zn catalyst present in the vitrimer. The macroscopic phase change of the material during the produc on/recycling cycle is shown in Erreur ! Source du renvoi introuvable.: the ini al liquid resin formula on is first cured and transformed into a vitrimer, the vitrimer is recycled as a viscous resin (which is soluble in acetone), the resin can be cured again with the same curing condi ons obtaining a solid, whose gel frac on was determined to be around 86 %, which is only slightly lower than the one observed for the ini al vitrimer (90 %). It is evident that the reprocessed sample is darker than the original network: this is likely to be due to thermal oxida on as explained in the literature [START_REF] Shi | Recyclable 3D Prin ng of Vitrimer Epoxy[END_REF]. Besides the visual observa ons, the recycled vitrimer was subjected to spectroscopic and thermal tes ng. The ATR-FT-IR spectrum of the original resin and of the resin a er transesterifica on (i.e., EGdissolved vitrimer) are compared in Figure 9A: as expected, the recycled product does not contain epoxy groups (no peak appears at 910-915 cm -1 ), while it is s ll exhibi ng the ester related peak at 1740 cm -1 . Therefore, this suggests that de-crosslinking is not occurring. However, an increase of the absorbance of the band at around 3400 cm -1 related to the hydroxyl groups can be noted: this could indicate the presence of remaining ethylene glycol.

The ATR-FT-IR spectra of the vitrimer freshly cured and the one cured a er recycling are reported in Figure 9B: interes ngly, one can no ce but negligible differences between the two products, thus the chemical integrity of the material upon recycling was confirmed. Concerning the thermal behaviour of the material a er recycling, no major difference can be observed both by DSC and TGA. When comparing the DSC spectra of the pris ne sample and that of the recycled one (Figure 10) same glass transi on temperatures are measured (Tg ≈ -13°C), and no exothermic peak is found in both curves (i.e., curing is complete). Similar conclusions come from TGA measurements: the degrada on for both the pris ne network and the recycled vitrimer are similar. The viscoelas c behaviour of the network a er recycling was also inves gated. As described above, the relaxa on me τ and the ac va on energy Ea of the recycled material were determined from the relaxa on curves given in Figure 11A and from the rela onship between τ and temperature (τ values at different temperatures are plo ed in Figure 11B). Data of the neat vitrimer and of the recycled one are compared in Table 2. As one can observe, relaxa on me is greater for the recycled vitrimer than for the ini al vitrimer. These changes might be due to the loss of catalyst during the recycling process or to side-reac ons, such as etherifica on, that are reducing the efficiency of the transesterifica on.

On the other hand, the ac va on energy was determined to be 110 kJ mol -1 , which is consistent with literature (J. Zheng et al. 2021) for transesterifica on mechanisms. However, it is greater than the one of freshly cured vitrimer (≈85 kJ mol -1 ): this could confirm the occurrence of compe ve reac ons, such as etherifica on and thermo-oxida on.

Vitrimer printing by LDM: addition of cellulosic powder

LDM is an AM method suitable for thermoplas cs but adopted for thermoset resins. For this class of materials a er the two main prin ng steps, extrusion and deposi on, a curing step must follow. Each prin ng step requires different viscoelas c features. For being extruded the resin should exhibit shearthinning behaviour, while for the deposi on phase, when the uncured resin must hold the shape, yield stress and s ffness must be maximized [START_REF] Bouzidi | 3D Printable Fully Biomass-Based Composite Using Poly(Furfuryl Alcohol) as Binder and Cellulose as a Filler[END_REF][START_REF] M'barki | Linking Rheology and Printability for Dense and Strong Ceramics by Direct Ink Wri ng[END_REF]. Guidelines for successful LDM prin ng based on the results obtained in a previous work [START_REF] Thibaut | Development of fibrous cellulosic materials for the produc on of bio-based 3D printed objects by extrusion[END_REF] are: strong shear thinning behaviour, apparent viscosity η from approx. 10 4 Pa s at 0.1 s -1 to 10 Pa s at 10² s -1 ; yield stress τy in the range 10 2 -10 3 Pa; s ffness G' between 10 4 and 10 5 Pa. Apparent viscosity measurement performed on the vitrimer precursors is shown in Figure 12A (black curve): it indicates that the epoxy resin and the acid hardener mixture does not sa sfy any of the above criteria. Therefore, we decided to add a rheology modifier: a cellulose powder was preferred being biobased, widely available and cheap. To minimize the risk of clogging of the extruder, the cellulosic filler had par cle size below 12 µm as it should not exceed 1/100 th to 10/100 th of the nozzle diameter of 0.7 mm [START_REF] Thibaut | Development of fibrous cellulosic materials for the produc on of bio-based 3D printed objects by extrusion[END_REF].

Viscosity curves were recorded for different contents of cellulose powder, from 30 to 42 wt% (Figure 12A). The apparent viscosity of the mixtures with a different content of cellulose varied from 2000 Pa s to 40000 Pa s at 0.1 s -1 and it was above 100 Pa s at 10 2 s -1 for any composi on. The flow curves were fi ed according to the Ostwald-De Waele power law (Eq. 5):

𝜂 = 𝐾𝛾̇

Eq. 5

where K is the consistency index and n is the thinning exponent. K is related to the viscosity at low shear rate, at rest, whereas n defines the rheologic behaviour of the material and when n = 0 the ideal shear-thinning behaviour is observed. In Figure 12B, K and n are plo ed as a func on of filler on. As expected, the higher the cellulose powder content, the higher the K value; n = 0.96 for the pure resin, then it diminishes and reaches a value of 0.1-0.3 when the cellulose content is 37-42 wt%. S ffness G' and yield stress τy were measured by the Amplitude Sweeps Test: the obtained curves are plo ed in Figure 13A. G' value was taken as the value of the storage modulus in the Linear Viscoelas c Region (LVER). τy, i.e., the minimum stress required for the resin to start flowing, was evaluated according to the Herschel-Bulkley model for a non-Newtonian fluid exhibi ng a yield stress τ0 (Eq. 6):

𝜏 = 𝜏 + 𝐾𝛾̇ Eq. 6
where K is the consistency index and n is the dimensionless flow index. The obtained values for the s ffness G' and the yield stress τy are reported in Figure 13BFigure 1: both are increasing as a func on of the filler content, as expected. With a content of at least 37 wt% of cellulose filler, both s ffness (G'= 3 10 5 Pa) and yield stress (τy = 150 Pa) are sa sfying the criteria of printability (i.e., τy in the range 10 2 -10 3 Pa and G' between 10 4 and 10 5 Pa). This on was therefore chosen for curing and further characteriza on in view of prin ng trials. The curing of the composite was performed in the same condi ons of the neat resin (200 °C, 6 h), and by ATR-FT-IR (Figure 14) the successful crosslinking of the vitrimer in the presence of filler was demonstrated. As for the pure epoxy resin and acid hardener, also in the presence of cellulose filler, the disappearance of the epoxide band at 910-915 cm -1 and the shi of the carbonyl band from 1715 cm -1 to a higher wavenumber, 1740 cm -1 , assessing the transforma on of the carboxylic group into the ester group, are observed with curing. As for the ini al resin, a broad peak at around 3400 cm -1 , which is related to the hydroxyl groups, can also be detected. This result is par cularly interes ng as the hydroxyls will be also used for the dynamic transesterifica on reac on. In the FT-IR spectra of Figure 14, one can clearly iden fy bands due to cellulose in the region around 1100 cm -1 .

Figure 14: ATR-FT-IR spectra of the uncured and cured composite with 37 wt% of cellulose filler, compared to the spectra of the cured vitrimer.

Successful crosslinking was also confirmed by the high value of the insoluble frac on (94 %). Main thermal proper es of the cured composite are listed in Table 3, and compared to those of the cured vitrimer. The Tg of the composite measured by DSC was found close to the one of the vitrimer, with a value around -15 °C. In the TGA curve of the composite, in addi on to the vitrimer degrada on, an early degrada on at 300 °C due to the cellulose powder can be detected. Therefore, the degrada on characteris c temperatures of the composite are lower than those of the neat vitrimer (Table 3). Residual masses at 800 °C for the composite are higher due to the presence of the filler. Preliminary tests on chemical recycling were performed also for the composite sample, applying the same procedure as for the neat vitrimer (reaction described in Figure 7). The ATR-FT-IR spectrum of the recycled composite had no noticeable difference with respect to the pristine composite (Figure in the Supporting Information). However, a change in Tg was measured with a shift from -15 °C to -21 °C after recycling. This suggests that with the recycling process a slightly different network can be obtained. Additionally, although the thermal degradation profiles were almost the same, small differences in the characteristic degradation temperatures are present, as reported in Table 3. The DSC and TGA thermograms can be found in Figure of the Supporting Information. Finally, the prin ng by LDM of the epoxy vitrimer containing 37 wt% of cellulose powder was a empted (see video). A er 3D prin ng, objects were thermally cured following the same thermal process applied for the neat vitrimer and its composite, i.e., hea ng at 200 °C for 6 h. As it can be observed in Figure 15 (top), the composite with 37 wt% of cellulose powder was successfully printed both in the form of a 20 x 20 mm 2 cube and in the form of a cylinder of 20 mm height and diameter of 20 mm. These results confirm that the composite vitrimer has the rheological proper es required for LDM, i.e., the vitrimer is suitable for an extrusion-based 3D prin ng once the rheology is tailored with the aid of a filler. A er the curing cycle, the shape of the composite pieces was almost retained as it is shown in Figure 15 (bo om): at the bo om the objects a certain deforma on is detectable, more evident for the cube. Also, some defects are present at the external surface due to the release of ace c acid from the catalyst and/or moisture from the cellulose powder. Overall, it is proved that the fully biobased vitrimer composite designed in this work is recyclable and printable by LDM. Although the prin ng tests are preliminary, these results are interes ng as they are in line with the current research trends for sustainability and with the perspec ves of research in addi ve manufacturing. In fact, it was recently pointed out that too few vitrimers are available for 3D prin ng [H. [START_REF] Zhang | Recycling strategies for vitrimers[END_REF], among which the biobased ones are rare. Moreover, in the specific field of LDM no example regarding vitrimers is reported yet. As men oned in the introduc on, with respect to other AM technologies suitable for vitrimers, i.e. stereolithography (SLA), digit light processing (DLP) and fused deposi on modeling (FDM), LDM is compe ve as it works at room temperature, needs a simple set up, and has good capabili es for producing large size pieces. Therefore, coupling a sustainable technology and a sustainable material is very promising.

Benchmarking further the obtained hybrid network against similar epoxy vitrimers, our polyestercellulose system requires similar temperatures and longer processing me for being reprocessed. At the same me, it has a relevant advantage versus its homologues: thanks to its viscoelas c proper es it requires neither a precrosslinking step (pre-curing) before prin ng nor hea ng during extrusion [START_REF] Shi | Recyclable 3D Prin ng of Vitrimer Epoxy[END_REF].

Conclusions

In this work, we proposed for the first me the processing of a fully biobased epoxy vitrimer composite by Liquid Deposi on Modelling (LDM) and proved the suitability of this simple AM technology for processing recyclable epoxy composites. The biobased sources of both the network precursors (i.e. the epoxide and the acid hardener) and the filler, the reversibility of the curing process and the sustainability of the processing technology, guarantee a much lower environmental impact compared to the fossil-based epoxides treated with tradi onal processing. First, we successfully produced and characterized a new Covalent Adaptable Network based on a transesterifica on mechanism, made of up to 98 % biobased compounds, i.e., a mul func onal epoxy resin derived from cardanol and a hardener with a 100% biobased content. The developed material had a Tg below room temperature (thus it is rubbery), a topology freezing temperature around 71°C but a quite high relaxa on me, i.e., 20 minutes at 200°C. The biobased vitrimer was successfully recycled and reprocessed thanks to a chemical recycling path, using ethylene glycol as a solvent. The recycled material maintained similar proper es of the pris ne vitrimer, with iden cal Tg (Tg ≈ -13°C) and unchanged thermal resistance as well as the viscoelas c proper es. The rheological behaviour of the vitrimer was not found suitable for LDM 3D prin ng and cellulose powder, a low-cost biobased filler, was added as a rheology modifier. Through a careful design of the viscoelas c proper es, a formula on at rela vely high cellulose content (37 wt%) was successfully printed without any pre-curing step and hea ng during extrusion. A er modelling, the curing assured the reten on of the shape of the printed pieces. The same process for recycling could be adopted for reprocessing the composite, allowing the recovery of the polymeric matrix and of the cellulose if needed.

CRediT authorship contribution statement

Figure 1 :

 1 Figure 1: Chemical structures of the biobased epoxide NC-514S and biobased hardener Pripol 1040.

Figure 2 :

 2 Figure 2: Esterification reaction between the epoxy resin and the acid, in the presence of zinc catalyst.

Figure 3 :

 3 Figure3: ATR-FT-IR spectra of the epoxy/acid system before (t=0 h) and after (t=6 h) curing.

Figure 4 :

 4 Figure 4: DSC thermograms of the epoxy/acid system before and after curing.

Figure 5 :

 5 Figure 5: TGA thermograms of the cured polyester network and of the pure epoxy resin.

Figure 6 :

 6 Figure 6: Viscoelasticity study of the cured vitrimer. A) Normalized stress relaxation curves at several temperatures. B) Plot of ln (𝜏) versus 1000/T.

Figure 7 :

 7 Figure 7: Chemical recycling route, adapted from (Shi et al. 2016).

Figure 8 :

 8 Figure 8: Production and recycling cycle of the biobased epoxy vitrimer.

Figure 9 :

 9 Figure 9: ATR-FT-IR spectroscopy analysis of the vitrimer, before and after chemical recycling. A) Spectra of uncured vitrimer and of EG-dissolved vitrimer. B) Spectra of cured vitrimer and cured recycled vitrimer.

Figure 10 :

 10 Figure 10: Thermal analysis of the vitrimer, before and after chemical recycling. A) DSC thermograms of cured vitrimer and of cured recycled vitrimer. B) TGA of cured vitrimer and of cured recycled vitrimer.

Figure 11 :

 11 Figure 11: Viscoelasticity study of the recycled vitrimer. A) Normalized stress relaxation curves at three different temperatures for the cured recycled vitrimer. B) Plot of ln (𝜏) versus 1000/T.

Figure 12 :

 12 Figure 12: Rheologic characterization of the vitrimer filled with different contents of cellulose powder. A) Apparent viscosity of the vitrimer and of a selection of composites. For the composites measurements stopped due to measuring issues at high shear rate, such as rip-off of samples. The experimental curves are fitted with the Ostwald-De Waele power law (Eq. 5). B) Consistency index and thinning exponent for the composites.

Figure 13 :

 13 Figure 13: Amplitude Sweep Test results of the epoxy vitrimer filled with different contents of cellulose powder. A) Storage and loss modulus obtained in Amplitude Sweep Test at 1 Hz. B) Stiffness G' and yield stress 𝜏 for composites with cellulose content ≥ 30 wt%.

Figure 15 :

 15 Figure 15: Picture of printed parts before (top) and after curing (bottom), made with the composite epoxy vitrimer with 37 wt% of cellulose filler.

Table 1 :

 1 TGA thermal degradation temperatures and residual mass at different temperatures.

	Material	𝑇 (°C)	𝑇 (°C)	𝑚 ° (%) Under N2	𝑚 ° (%) Under air
	Cured vitrimer	319	443	6	3
	NC-514S	296	408	3	0

Table 2 :

 2 Relaxation time of cured vitrimer and cured recycled vitrimer at different temperature values.

	Temperature (°C)	Cured vitrimer	Relaxa on me (s) Cured recycled vitrimer
	200	≈1200	≈1800
	180	≈3000	≈6300
	160	≈10000	≈26800*
	*Projected value

Table 3 :

 3 Tg values by DSC, and TGA thermal degradation temperatures and residual mass at different temperature for cured vitrimer, cured composite and cured recycled composite samples.

	Material	Tg (°C)	𝑇 (°C)	𝑇 (°C)	𝑚 ° (%) Under N2
	Cured vitrimer	-13	319	443	6
	Cured composite	-15	290	423	13
	Cured recycled composite	-21	297	414	9

Data availability Statement

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

Declaration of competing interest

The authors declare that they have no known compe ng financial interests or personal rela onship that could have appeared to influence the work reported in this paper.

Suppor ng informa on

Calcula on of Tv

As reported in previous protocol reported by [START_REF] Tretbar | Direct Silyl Ether Metathesis for Vitrimers with Excep onal Thermal Stability[END_REF], the topology freezing temperature Tv can be determined combining DMA and stress relaxa on experiments. This transi on is considered to be when viscosity reach 10 12 Pa.s. Using Maxwell equa on:

With 𝐺 =

We can plug the extrapolated * into equa on (3) which gives us the value of Tv to be 71°C.

Calcula on of the crosslinking density of the Cardolite-Pripol polyester

According to the theory of rubber elas city the crosslink density can be calculated according to

𝜈 =

(4) E' is the storage modulus at T, 𝑇 is the temperature at the rubbery plateau, determined to be Tg + 50, i.e., 𝑇 = 310.15 K as from DSC measurement the biobased vitrimer has Tg = -13°C. From the DMA plot, at the temperature 𝑇, the storage modulus is E'=1800000 Pa. Given the gas constant R=8.314J/mol.K, the crosslink density is determined to be 𝜈 = 233 mol/m 3 .


Dr Didier Dupont
email: didier.dupont@rennes.inra.fr

Giuseppina Mandalari

Daniel Mollé

Julien Jardin

Joëlle Léonil

Richard M Faulks

Martin S J Wickham

E N Clare Mills

Alan R Mackie

Daniel Molle

Comparative resistance of food proteins to adult and infant in vitro digestion models

Keywords: beta-lactoglobulin, β-CN, β-casein, OVA, ovalbumin, PC, phosphatidylcholine, SGF, Simulated Gastric Fluid milk, egg, food allergy, phosphatidylcholine, in vitro digestion

des établissements d'enseignement et de recherche

Introduction

Understanding the fate of dietary proteins during gastrointestinal digestion has become of particular interest because of the potential role digestion may play in determining the allergenic potential of foods [START_REF] Mills | Structural, biological and evolutionary relationships of plant food allergens sensitising via the gastrointestinal tract[END_REF]. One important allergenic food is cows' milk, which in industrialised nations is estimated to affect approximately 2% of infants under 2 years of age [START_REF] Sampson | Food allergy. Part 1: immunopathogenesis and clinical disorders[END_REF][START_REF] Hill | Clinical spectrum of food allergy in children in Australia and South-East Asia: identification and targets for treatment[END_REF]. The major allergenic fraction of cow's milk is the casein fraction, comprising a mixture of s 1 -, s 2 -, -and casein (Bos d8). Despite their excellent digestibility, caseins are generally considered to be potent allergens [START_REF] Wal | Structure and function of milk allergens[END_REF]. The globular protein -lactoglobulin (-Lg, Bos d5), present in the whey fraction of the milk of most mammals, is also considered to be a major cause of allergic response to cows' milk in humans [START_REF] Mantyjarvi | Lipocalins as allergens[END_REF]. A second important allergenic food infancy and early childhood is hens' egg [START_REF] Rona | The prevalence of food allergy: A meta-analysis[END_REF]. Almost all the allergens of egg are derived from the white and include the proteins ovomucoid, ovalbumin and lysozyme [START_REF] Vieths | EuroPrevall food allergen library[END_REF]. Certain of these, such as ovalbumin (Gal d2), are characteristically more resistant to gastrointestinal digestion as a consequence of the glycosylation and globular structure of this protein.

It may be that, whilst the bulk of food proteins are broken down into immunologically inactive fragments, very small proportions of immunologically-active material may escape digestion. The extent of this digestion may also be affected by factors such as the maturity of the gut and its healthy functioning [START_REF] Untersmayer | The role of protein digestibility and antacids on food allergy outcomes[END_REF] and raised intragastric pH caused by anti-ulcer medication, as has been shown for studies of fish allergens [START_REF] Untersmayer | The effects of gastric digestion on codfish allergenicity[END_REF]. Indeed, poor digestion of codfish represents a risk factor for anaphylaxis in patients with allergy [START_REF] Untersmayer | Incomplete digestion of codfish represents a risk factor for anaphylaxis in patients with allergy[END_REF].

Furthermore, it has been shown that cows' milk whey peptides need to have a molecular weight greater than 3000 daltons (around 25 residues) in order to stimulate an immune response [START_REF] Bannon | Protein digestibility and relevance to allergenicity[END_REF], and an allergen must contain at least two IgE epitopes, each of which with a minimum of 15 amino acid residues long, in order to make the antibody binding possible [START_REF] Van Beresteijn | Residual antigenicity of hypoallergenic infant formulas and the occurrence of milkspecific IgE antibodies in patients with clinical allergy[END_REF]. Fragments combining multiple epitopes at least 1nm in size are required to elicit histamine release [START_REF] Kane | Cross-linking of IgE-receptor complexes by rigid bivalent antigens greater than 200A in length triggers cellular degranulation[END_REF][START_REF] Schöll | Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice[END_REF]. Thus, large stable fragments, as well as intact proteins, have the potential to trigger both initial sensitisation and phases of allergic responses.

Since cows' milk and hens' egg allergies are diseases of infancy, in vitro studies of digestion, even using systems which seek to be physiologically-relevant, have not, in the past, taken into account the conditions found in the immature gastrointestinal tract of infants. Whilst the gold standard for investigating the human digestive process are in vivo approaches these are constrained for ethical and practical reasons, especially when considering infants. There is therefore a need to develop effective in vitro models of digestion which mirror the conditions and processes that actually occur in vivo. Several more or less refined models of the biochemical conditions found in adults have been used to study food protein digestion [START_REF] Vieths | Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure[END_REF][START_REF] Mouecoucou | lactoglobulin/polysaccharide interactions during in vitro gastric and pancreatic hydrolysis assessed in dialysis bags of different molecular weight cut-offs[END_REF][START_REF] Moreno | Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion[END_REF][START_REF] Jin | Moving toward a more physiological model: application of mucine to refine the in vitro digestion/Caco-2 cell culture system[END_REF]. Based on published data a prototype biochemical model of infant gastrointestinal digestion was developed and used to assess the digestion of model cows' milk allergens -Lg, -casein (β-CN) and the archetypal model allergenic protein from hens' egg, ovalbumin (OVA). One of these, -CN, is extensively degraded during digestion [START_REF] Astwood | Stability of food allergens to digestion in vitro[END_REF] whilst the others are both more resistant [START_REF] Astwood | Stability of food allergens to digestion in vitro[END_REF][START_REF] Thomas | A multilaboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins[END_REF]. In addition to using a biochemical assessment of digestibility, including immunoreactivity of residual proteins and peptides resulting from digestion by a panel of relevant monoclonal and polyclonal antibodies [START_REF] Johansson | Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: The case of casein micelle[END_REF], we have undertaken a comprehensive mapping of proteolysis products resulting from digestion of -CN using mass spectrometry.

Materials and methods

Chemicals

Unless otherwise stated, chemicals were from commercial origin (Sigma, St-Louis, MO, USA). Purity of β-Lg, β-CN and OVA was ≥ 90%.

In vitro adult digestion model

Phospholipid vesicles were prepared using a modification of a procedure described previously [START_REF] Mandalari | Release of protein, lipid and vitamin E from almond seeds during digestion[END_REF]. Solvent was removed from a 0.94 mL aliquot of L-phosphatidylcholine 63.5 mM in chloroform stock solution (PC, egg lecithin grade 1, 99% purity, purchased from Lipid Products South Nutfield, Surrey, UK) and dried under rotary evaporation at 5°C in order to make a thin film of phospholipids. The film of phospholipids was then suspended in 12.2 mL of warmed simulated gastric fluid (SGF, 0.15 M NaCl, pH 2.5) and the suspension sonicated for 7 min using a sonication probe (Status US 200, Avestin Inc., Canada) at 5°C in a coolant-jacketed vessel. The liposomes were then covered with a cushion of argon before equilibrating at 37°C for 20 min in a shaking incubator. Proteolysis was performed essentially as previously described [START_REF] Mandalari | Release of protein, lipid and vitamin E from almond seeds during digestion[END_REF] using triplicate incubations at 37°C. Briefly, β-Lg, β-CN or OVA were dissolved in SGF (1 mg/mL), mixed with PC vesicles and the pH was adjusted to 2.5 with 0.5 M HCl solution. Porcine gastric mucosa pepsin (EC 3.4.23.1, Sigma, activity: 3,300 U/mg of protein calculated using haemoglobin as a substrate) was added to give 182 U of pepsin/mg of β-Lg, β-CN or OVA (0.05 mM, final concentration). Aliquots (100 µL) were removed over the 60 min digestion time course. Pepsinolysis was stopped by raising the pH to 7.0 using 0.5 M ammonium bicarbonate (BDH, Pole, Dorset, UK). Prior to duodenal proteolysis, the pH of samples was adjusted to 6.5 by addition of 0.1 M NaOH and duodenal digestion components added to give final concentrations as follows: 4 mM sodium taurocholate, 4 mM sodium glycodeoxycholate, 26.1 mM Bis-Tris buffer pH 6.5, 0.4 U/mg of β-Lg, β-CN or OVA bovine -chymotrypsin (activity 40 U/mg of protein using benzoyltyrosine ethyl ester, BTEE, as substrate), 34.5 U/mg of β-Lg, β-CN or OVA porcine trypsin (activity 13,800 U/mg of protein using benzoylarginine ethyl ester, BAEE, as substrate). Aliquots (100 µL) were removed over the 30 min digestion time course, and proteolysis stopped by addition of a two-fold excess of soybean Bowmann-Birk trypsin-chymotrypsin inhibitor above that calculated to inhibit trypsin and chymotrypsin in the digestion mix.

In vitro infant digestion model

The infant digestion model was applied to the same batches of -Lg, -CN and OVA using the same protocol as described above but with the following modifications:

1) pH of the gastric digestion mix was adjusted at 3.0 instead of 2.5;

2) the pepsin concentration in the gastric digestion mix was decreased by a factor of 8;

3) the duodenal digestion mix was altered by reducing the bile salt concentration by a factor of 4, whilst the PC, trypsin and chymotrypsin concentrations were reduced by a factor of 10.

As a consequence of the reduced levels of trypsin and chymotrypsin a 10-fold lower concentration of Bowmann-Birk inhibitor was added to terminate simulated duodenal proteolysis.

Antibodies

Eight mouse monoclonal antibodies specific for -CN were taken from INRA's collection [START_REF] Johansson | Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: The case of casein micelle[END_REF] and were directed against the following fragment of this protein: -CN (f1-25), (f42-56) for two antibodies, (f76-93), (f133-150) for two antibodies, (f167-178) and (f184-202).

Rabbit polyclonal antibodies specific for -Lg and -CN were raised following the protocol previously described by [START_REF] Senocq | Monoclonal antibodies against bovine β-casein: production and epitope characterization[END_REF] [START_REF] Senocq | Monoclonal antibodies against bovine β-casein: production and epitope characterization[END_REF]. OVA specific rabbit polyclonal antibodies were kindly provided by Dr. Didier Levieux (INRA, Theix, France).

SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 10 % polyacrylamide NuPAGE Novex Bis-Tris precast gels (Invitrogen, Carlsbad, CA, USA) according to the manufacturer instructions. All samples were reduced using 0.5 M DL-dithiothreitol prior to analysis. Ten micrograms of each allergen were loaded onto each lane. Gels were fixed in 50% (v/v) methanol, 10% (v/v) acetic acid and after 30 min were rinsed in deionised water prior to staining with Colloidal Blue Staining Kit (Invitrogen Carlsbad, CA, USA). Image analysis of SDS-PAGE gels was carried out using Totallab 120 (Nonlinear Dynamics Ltd, Newcastle, UK) using an automated process supplemented with occasional manual adjustments. Each lane was first defined, and the average intensity across the width of these lanes depicted as a function of the distance in pixel from the top of the image. The background was subtracted from each profile (parameters adjusted on a case by case basis), after which the bands were identified. Gaussian curves were fitted to the bands, which allowed overlapping bands to be deconvoluted. The lanes of molecular weight standards were then used to reference the position of the bands. For this, lanes corresponding to a given Rf or MW were drawn across the width of the gel, allowing the position of each band to then be estimated. The output of this process was a list of bands defined either by their Rf or by their MW, together with an estimate of the volume under the curve corresponding to each band (volume of fitted Gaussian curve).

Reverse Phase-HPLC

Samples (25 l) were loaded onto a Phenomenex (Torrance, CA, USA) Jupiter Proteo 90 Å pore size, 4 m particle size (250 x 4.6 mm i.d.) column attached to a Dionex HPLC system (Dionex Co., Amsterdam, The Netherlands) with a diode array detector. Samples were eluted using 0.1% (w/v) trifluoroacetic acid in ultra-pure water as solvent A and 0.085% (w/v) trifluoroacetic acid in ultra-pure water/acetonitrile (10:90, v/v) as solvent B following the method described by Moreno et al. [START_REF] Moreno | Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa)[END_REF].

Inhibition ELISA

Inhibition ELISA using -Lg, -CN and OVA specific polyclonal antibodies was applied to the samples collected throughout digestion of these 3 proteins in order to determine the residual immunoreactivity of each protein during the digestive process.

ELISA plates (NUNC, Maxisorp, Roskilde, Denmark) were coated with 0.5 µg/mL -Lg, -CN or OVA in 0.1 M bicarbonate buffer, pH 9.6 (100 µL per well) and incubated for 1 h at 37°C. Wells were rinsed between incubation steps for 15 s with four changes of 250 µL phosphate-buffered saline, 0.05% Tween 20 (PBS-T, Sigma) using a Model 1575 Immunowash microplate washer (Bio-Rad, Hercules, CA, USA).

Blocking of the remaining binding sites was performed with 250 µL fish gelatin (Sigma, St-Louis, MO, USA) at 10 g/L in PBS-T for 1 h at 37°C. Serial dilutions of -Lg, -CN or OVA in PBS-T were used as standards (concentrations ranging from 0 to 100 µg/mL). Samples diluted in PBS-T (four dilutions from 1:1000 to 1:5000, 75 µL) were incubated in test tubes with 75 µL of rabbit polyclonal antibodies specific for -Lg, -CN or OVA diluted at 1:32000, 1:3500 and 1:5000 (v:v) respectively and incubated for 1 h at 37°C. One hundred microlitres of the mixture was then added to each ELISA plate well and further incubated for 1 h at 37°C. The reaction was revealed by incubating 100 µL of goat anti-rabbit Ig alkaline phosphatase conjugate (Sigma, St-Louis, MO, USA) diluted 1:3000 (v:v) in PBS-T for 1 h at 37°C. Finally, 100 µL p-nitrophenyl phosphate (Sigma, St-Louis, MO, USA) at 1 g/L 1 M diethanolamine-HCl, 1 mM MgCl 2 , 0.1 mM zinc acetate were incubated in the wells.

After 30 min at 37°C, the absorbance at 405 nm was read against a blank (substrate only) using a Benchmark Plus microplate spectrophotometer (Bio-Rad, Hercules, CA, USA).

Indirect ELISA

This method was used to detect the -CN regions resistant to digestion using a panel of 8 monoclonal antibodies (Mabs) specific for fragments as follows: -CN (f1-25) for 1 Mab, (f42-56) for 2 Mabs, (f76-93), (f133-150) for 2 Mabs, (f167-178) for 1 Mab, (f184-202) for 1 Mab. Briefly, 100 µL of adult or infant -CN digest diluted 1:1000 (v:v) in 0.1 M bicarbonate buffer, pH 9.6 were coated onto a micro-titre plate (NUNC, Maxisorp, Roskilde, Denmark) and incubated 1 h at 37°C. Wells were rinsed between incubation steps for 15 s with four changes of 250 µL PBS-T using a Model 1575 Immunowash microplate washer (Bio-Rad, Hercules, CA, USA). The remaining binding sites were blocked by incubating 250 µL fish gelatin (Sigma, St-Louis, MO, USA) at 10 g/L PBS-T for 1 h at 37°C. Hybridoma culture supernatants were diluted 1:2 (v:v) in PBS-T and incubated for 1 h at 37°C. Bound mouse Ig was detected by incubating 100 µL of goat anti-mouse Ig alkaline phosphatase conjugate (Sigma, St-Louis, MO, USA) diluted 1:3000 in PBS-T for 1 h at 37°C. Following the last rinsing, 100 µL p-nitrophenyl phosphate (Sigma, St-Louis, MO, USA) at 1 g/L in 1 M diethanolamine-HCl, 1 mM MgCl 2 , 0.1 mM zinc acetate were added to each well.

After 30 min at 37°C, the absorbance at 405 nm was read against a blank (substrate only) and corrected according to the background signal in the absence of antigen using a Benchmark Plus microplate spectrophotometer (Bio-Rad, Hercules, CA, USA).

Immunoblotting

The samples collected at the end of the digestion process were electrophoresed as described above. Immediately after separation, proteins and peptides were transferred onto a 0.2 m pore size nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) at 15 V and 400 mA during 20 min using a semi-dry transfer cell (Trans-Blot SD, Bio-Rad, Hercules, CA, USA) and a 39 mM glycine, 48 mM Tris, 0.0375% (w:v) sodium dodecyl sulfate and 20% (v:v) methanol transfer buffer. Immunodetection was conducted according to the following procedure. The membrane was incubated at room temperature for 1 h periods in PBS-T, with, successively, 1% (w:v) gelatine, -Lg, -CN or OVA specific rabbit polyclonal antibodies at 1:2000, 1:500, and 1:1000 (v:v) respectively and goat anti-rabbit immunoglobulin alkaline phosphatise conjugate at 1:500 (v:v). The membrane was washed between each step by soaking for five min in four changes of the same buffer. After the last washing, the membrane was stained with Fast 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium tablets (Sigma, St-Louis, MO, USA) at 1 tablet in 10 mL water.

Nano-Liquid Chromatography/Tandem Mass Spectrometry (LC-MS-MS)

Samples collected at the end of duodenal digestion in both adult and infant models were analysed by LC-MS-MS in order to identify the peptides remaining after

Results

Development of a prototype biochemical model of infant gastrointestinal digestion

An adult in vitro model of gastroduodenal digestion has been described previously which was based on in vivo data obtained by gastric and duodenal aspiration and from collection of effluent from ileostomy volunteers at the Institute of Food Research [START_REF] Moreno | Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion[END_REF][START_REF] Mandalari | Release of protein, lipid and vitamin E from almond seeds during digestion[END_REF]. This was modified according to the published data [START_REF]Scientific tables[END_REF][START_REF] Hamosh | Digestion in the newborn[END_REF][START_REF] Walthall | Postnatal development of the gastrointestinal system: a species comparison[END_REF] to provide conditions that mimicked more closely those found in the infant gut. More precisely, gastric phase digestion was undertaken at pH 3.0 instead of 2.5 in the adult model because it corresponds to the gastric fasting pH as it was recorded in mature [START_REF] Mitchell | Simultaneous monitoring of gastric and oesophageal pH reveals limitations of conventional oesophageal pH monitoring in milk fed infants[END_REF] and preterm infants [START_REF] Armand | Effect of human milk or formula on gastric function and fat digestion in the premature[END_REF][START_REF] Omari | Multipoint measurement of intragastric pH in healthy preterm infants[END_REF][START_REF] Lopez-Alonso | Twenty-fourhour oesophageal impedance-pH monitoring in healthy preterm neonates: rate and characteristics of acid, weakly acidic and weakly alkaline gastroesophageal reflux[END_REF]. Pepsin, trypsin and chymotrypsin concentrations were decreased by a factor of 8, 10 and 10 respectively compared to the adult model [START_REF] Dipalma | Lipase and pepsin activity in the gastric mucosa of infants, children, and adults[END_REF][START_REF] Henderson | Bioactive Components of Human Milk -Gastric proteolysis in preterm infants fed mother's milk or formula[END_REF]. The concentration of the gastric surfactant, phosphatidylcholine, was lowered by a factor of 10 and bile salts, consisting of a mixture of equimolar quantities (0.0125M) of sodium taurocholate and glyco-deoxycholic acid, was reduced 4-fold compared to the adult model [START_REF]Scientific tables[END_REF].

Comparison of infant and adult models on kinetics of digestion

In general the proteins were digested in a similar manner using both the adult and infant model, but as might be anticipated given the lower protein: protease ratios employed the kinetics of digestion were slower for the infant model. Thus, -Lg, a protein known to be highly resistant to pepsinolysis [START_REF] Schmidt | Raising the pH of the pepsin-catalysed hydrolysis of bovine whey proteins increases the antigenicity of the hydrolysates[END_REF], was not affected during gastric digestion, being virtually unaltered after 60 min under both the adult and the infant gastro-duodenal models (results not shown). The duodenal phase showed differences between the two gut models, -Lg being more extensively degraded in the infant model with only ~ 56% of the intact protein remaining after 30 min duodenal digestion compared with ~ 72% remaining at the same time point in the adult model (Fig. 1a,b; Fig. 2a). This was surprising since the concentration of trypsin and chymotrypsin in the infant model was reduced by a factor of 10 compared to the adult model. This observation can be explained by the fact that the gastric PC concentration was also lowered in the infant model, reducing the protective effect of PC normally observed in the adult model [START_REF] Mandalari | Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis[END_REF]. The kinetics of appearance of digestion products was also different between the adult and the infant models. As previously reported [START_REF] Mandalari | Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis[END_REF], the Mr ~12900 and ~11400 Da polypeptides were observed early during digestion in the presence of physiological concentrations of PC, and persisted until the end of duodenal digestion. However, Mr ~12900 Da polypeptide was not present in the infant gut model, indicating a different pathway of degradation.

The appearance of a Mr ~ 7000 Da polypeptide, along with the poorly resolved Mr ~ 3700 Da polypeptide, was only observed in the adult model, both fragments being evident after 30 min digestion.

In contrast, -CN was hydrolysed rapidly in both infant and adult models, the Mr 24000 Da polypeptide corresponding to the intact protein not being detected after 10 min gastric digestion (Fig. 1c,d

Evolution of the protein immunoreactivity during digestion

The residual immunoreactivity of -Lg, -CN and OVA was determined, throughout digestion, by inhibition ELISA using polyclonal antisera raised to the intact proteins (Fig. 3). The immunoreactivity of -Lg was unaltered by gastric digestion in both models, more than 94% of the residual immunoreactivity being retained after 60 min gastric digestion (Fig. 3a). The duodenal phase resulted in a decrease of -Lg immunoreactivity which was more significant in the infant model (47.3% at the end of digestion) than in the adult one (68.3% remaining after digestion). The decrease in -Lg immunoreactivity occurred during the first two minutes of duodenal digestion using the infant model whereas it was more gradual in the adult model. -CN was extremely rapidly hydrolysed by the two models, its residual immunoreactivity being reduced to 8.1% of the starting intact protein after 2 min gastric digestion with the infant model and being completely lost after 20 min gastric digestion with the adult model (Fig. 3b). Surprisingly the -CN immunoreactivity was not abolished following digestion with the infant model and even increased at the end of the gastric/ beginning of the duodenal phases to reach 20% of the intact protein.

This was a consistent observation in replicate digestion experiments, for which we currently lack an explanation. However, the concomitant presence of Mr ~ 7400 Da and a Mr ~ 4400 Da polypeptides as observed by SDS-PAGE persisting for up to 60 min in the infant model of digestion, tend to confirm the resistance of β-CN fragments throughout the gastric phase. Finally the immunoreactivity decreased during the duodenal digestion in the infant model such that only 2.7% of -CN immunoreactivity remained at the end of the gastroduodenal process.

OVA immunoreactivity gradually decreased during both gastric and duodenal digestion with both infant and adult models but was more extensively degraded with the latter (Fig. 3c). Indeed, the OVA residual immunoreactivity was 70.6% and 21.1% at the end of digestion with the infant and adult models respectively.

Immunoblotting confirmed the differences in resistance to digestion of the 3 model proteins (Fig. 4) and showed that the anti--Lg and anti-OVA antibody preparations both recognised a spectrum of polypeptides resulting from digestion.

Thus, undigested -Lg was recognised, as indicated by the two major bands stained at Mr 18 and 36 kDa corresponding to monomeric and dimeric forms of this protein. These data support the observation by SDS-PAGE and ELISA that -Lg was more extensively degraded during the duodenal stage by the infant compared to the adult model, in contrast to OVA which showed a more extensive proteolysis with the adult than with the infant model, whilst -CN proved to be the most rapidly degraded protein in both digestion models. The changes in immunoreactivity do not precisely mirror the loss of intact protein, suggesting that the immunoreactivity may be influenced by changes in protein structure caused by partial proteolysis, recognition of peptides and peptide aggregates, as well as intact protein.

Identification of the area of resistance in -CN using specific monoclonal antibodies

Since -CN was extensively degraded to low Mr <3 kDa peptides a panel of 8 Mabs of known specificity able to recognise short segments of polypeptide sequences were used to map the residual peptides in the -CN digests (Fig. 5). With the adult model, peptides corresponding to residues 76-93 were recognised by Mab (f76-93) and survived gastric digestions with of the immunoreactivity of 70.8% -CN remaining.

Peptides corresponding to residues 1-25 and 167-178 returned 8.7% and 10.3% of the -CN immunoreactivity, as detected by the relevant Mab probes. At the end of duodenal digestion peptides corresponding to residues 76-93 were further degraded.

A slight residual immunoreactivity (7.9% of that of intact -CN) was observed for peptides corresponding to residues 167-178, which did not seem to be affected by duodenal digestion. These data suggest that hydrolysis of -CN in the infant model was less extensive, with residual immunoreactivity after gastric digestion being identified for fragments corresponding to residues 76-93 (52.3%), 133-150 (44.8-47%), 42-56 (19-19.4%), 1-25 (12.7%), 167-178 (9.6%) and 184-202 (8%) respectively. After the duodenal digestion peptides corresponding to residues 76-93 showed the highest residual immunoreactivity of 10.7%.

Identification of low molecular weight peptides in digests by LC-MS-MS

The method of choice for mapping the evolution of complex mixtures of peptides is LC-MS-MS. Thus, digested samples of the three model proteins used in this study were subjected to such analysis to allow the identification of short peptides with molecular masses between 450 and 2500 Da. Fig. 6 shows a typical example of the total ionisation current profile observed when digested β-CN was analysed (a), corresponding MS spectrum at retention times between 33.125 and 32.453 min (b), MS-MS spectrum of the ion 796.6 (c). Only the peptides identified unambiguously in digested β-Lg, β-CN and OVA are indicated in Fig. 7a,b,c respectively. The limitations of the technique mean that large peptides that are resistant to proteolysis during digestion will not ionise or fragment during LC-MS-MS and so could not be identified.

For -Lg digested using the adult model, 37 peptides were identified with molecular masses ranging from 472 to 1315 Da covering most of the sequence (Fig. 7a). Among those, 36 were found common to both variants A and B, whereas peptide corresponding to 110 SAEPEQSLV 118 originated only from variant A. This suggests that the replacement of an alanine residue (variant B) by a valine (variant A) resulted in the appearance of an extra peptide in the digested samples. Among the 37 peptides corresponding to fragments of -Lg A, 7 showed an oxidized methionine residue. Oxidation of methionine residues is observed very often in LC-MS-MS when samples are kept in the presence of oxygen prior to analysis (data not shown) and does not necessarily correspond to a biological oxidation of the residues during in vitro digestion. In -Lg digests obtained with the infant model, 36 peptides with molecular masses ranging from 545 to 1191 Da were identified unambiguously in both variants, 4 showing an oxidized methionine residue. Of the 37 peptides identified in the adult digest, 19 were found specifically in the adult and not in the infant digest. Most of the peptides with the highest molecular masses were specific to the model used to prepare them and probably relates to the protective effect of PC [START_REF] Mandalari | Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis[END_REF].

For -CN, 70 peptides with molecular masses ranging from 561 to 1730 Da were identified in the adult digest (Fig. 7b). Among these, a single oxidised methionine residue was found in 11 peptides, whilst a further 2 peptides had two oxidized methionine residues. In the infant digest, only 48 peptides with molecular masses ranging from 567 to 1451 Da were identified. Twenty nine peptides were common to both the adult and infant digests.

Maybe as a consequence of its glycosylated state only 20 peptides with molecular masses ranging from 514 to 1109 Da could be identified in the OVA adult digest (with 2 peptides and 1 peptides showing 1 and 2 oxidized methionine residues respectively; Fig. 7c) whereas 11 peptides with molecular masses ranging from 514 to 1161 Da were found in the infant digest (with 1 peptide having 1 oxidized methionine). Seven peptides were found identically in both infant and adult digests.

Tables 1-3 summarize the trypsin and chymotrypsin typical cuts that generated specific identified peptides from β-Lg, β-CN and OVA respectively. Differences in digestion products obtained between adult and infant models result from differences in both trypsin and chymotrypsin cleavages. The generation of peptide products by these proteases used in combination is complex, as in the relationship between parent-daughter peptides where two related peptides may actually originate from different peptides. In many instances predicted cleavages could not be experimentally verified. There are several hypotheses that could explain the absence of such cleavages. It can be due to a limited access of the area for the proteases because of steric hindrance, or that some of the peptides have not been detected in digested samples by LC-MS-MS because of their low concentration. In addition we have previously found that currently available tools for predicting pepsin cleavage are unreliable [START_REF] Rigby | Pepsinolysis products of the major peanut allergen Ara h 1 are IgE reactive[END_REF]. Although the present results are difficult to compare with previous studies because of differences in the digestion conditions (pH, enzyme/substrate ratio) some similarities can be emphasized.

Discussion

It had been anticipated that the lower concentrations of proteases used in the infant model would systematically result in less extensive degradation of the three model proteins used in this study -Lg, -CN and OVA. Whilst this was so for -CN and OVA, in this study -Lg was more extensively degraded by the infant than the adult digestion model. This is probably a result of the reduction in the protective effect that gastric PC has on native -Lg retarding duodenal digestion by trypsin and chymotrypsin, which is not observed for β-CN [START_REF] Mandalari | Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis[END_REF]. The protective effect of PC is dose-dependent and the reduction of PC in the infant model to give a 0.6:1 molar ratio of PC: protein affected the kinetics of digestion. We have recently shown that ~90% of β-Lg was degraded by trypsin and chymotrypsin at a 1:1 molar ratio of PC to protein in the adult gut model [START_REF] Mandalari | Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis[END_REF]. This indicates that the lower concentrations of duodenal proteases in the infant gut model together with the PC affected β-Lg digestion. The significance of this difference regarding digestion of milk proteins will relate to the effectiveness of any thermal processing used to treat milk in denaturing -Lg since thermal denaturation abolishes the protective effect of PC [START_REF] Mandalari | Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis[END_REF].

OVA was found to be more extensively hydrolysed during digestion using the adult compared the infant model. However, after both processes some large fragments remain uncleaved. Since a relatively low number of short peptides were identified in OVA digested samples by mass spectrometry, it suggests that the large fragments generated by in vitro digestion retained sufficient structure to allow them to resist to further digestion by proteases and thus renders them inaccessible to identification using this mass spectrometry technique. This may be in part due to the presence of N-glycans linked at Asn-292 which represent up to 3% of the protein by weight. These glycans, coupled with the presence of an intramolecular disulphide bond and two phosphorylation sites at residues 68 and 344 may both reduce protease accessibility to potential cleavage sites and will affect the ability of any peptides to ionise and fragment during LC-MS-MS.

-CN was extensively degraded during the gastric phase in both models of digestion, an observation consistent with the fact that pepsin has a preference for mobile poorly structured polypeptides, such as casein [START_REF] Astwood | Stability of food allergens to digestion in vitro[END_REF]. However, only the adult model resulted in a total disappearance of the protein whereas presence of a low level of resistant peptides which persisted throughout digestion with the infant model.

The region corresponding to residues 76-93 was shown to be particularly persistent.

A hydrophobicity profile of -CN shows that this area is very hydrophobic at pH 3.0 (Fig. 8) a factor which may contribute to its resistance to pepsinolysis either through changes in conformation or aggregation. At the beginning of the duodenal phase, when the pH is raised to 6.5, this area will become more hydrophilic and more accessible for proteases. It has 3 potential cleavage sites for chymotrypsin residues (77, 87 and 88) and our data confirmed it was cleaved by this protease. The region corresponding to residues 133-150 is also very hydrophobic at pH 3.0 and it may be protected from pepsinolysis for similar reasons to residues 76-93. Both these regions are contained within a 6 kDa peptide corresponding to residues 94-149 which has been found to resist pepsinolysis when presented in an emulsified form further supporting the role for hydrophobic interactions in protecting regions from pepsinolysis [START_REF] Macierzanka | Emulsification alters simulated gastrointestinal proteolysis of β-casein and βlactoglobulin[END_REF].

Mass spectrometry analysis also showed that fragments of β-CN with molecular masses up to 1451 Da (β-CN (f81-93)) could be found after simulated gastroduodenal digestion. It is interesting to note that peptide β-CN (f83-92), that is contained within this sequence has been reported as one of the major serum IgEbinding epitopes of β-CN in a panel of milk-allergic children [START_REF] Chatchatee | Identification of IgE and IgG binding epitopes on β-and k-casein in cow's milk allergic patients[END_REF]. Studies of allergenicity of the readily digestible allergen of peanut, Ara h 1, have indicated that aggregation of peptides may play a role in retaining allergenic activity [START_REF] Eiwegger | Gastroduodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential[END_REF]. It is possible that such phenomena would also apply to casein peptides and could explain the apparent increase in immunoreactivity of casein digests observed with a polyclonal anti β-CN antiserum at the end of the gastric and the beginning of the duodenal phases in the infant model.

For -lg, the major IgE epitopes reported in the literature correspond to areas where peptides resistant to digestion were identified by LC-MS-MS in the present study, i.e. 47-60, 67-78, 75-86, 127-140, 141-152 [START_REF] Cocco | Mutational analysis of immunoglobulin E-binding epitopes of -casein and -lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera[END_REF] and 75-84, 141-150 [START_REF] Järvinen | B-cell epitopes as a screening instrument for persistent cow's milk allergy[END_REF].

Finally, some of the peptides identified in the digested OVA sample like 53-60, 77-84, 178-187, 242-252, 251-259, 275-280, 322-343 have been reported as carrying IgE epitopes [START_REF] Mine | Epitope characterization of ovalbumin in BALB/c mice using different entry routes[END_REF][START_REF] Lopez-Exposito | Changes in the ovalbumin proteolysis profile by high pressure and its effect on IgG and IgE binding[END_REF].

In conclusion, we report here the development of an infant gut model for studying the in vitro digestion of purified food proteins. This model is static and pH is kept constant during the whole digestion process which might not perfectly reflect what happens during the digestion of complex foods. Indeed, it has been shown that the buffering capacity of food, particularly milk, causes a significant increase in pH during the gastric phase that will probably lower the enzyme activities [START_REF] Mitchell | Simultaneous monitoring of gastric and oesophageal pH reveals limitations of conventional oesophageal pH monitoring in milk fed infants[END_REF][START_REF] Armand | Effect of human milk or formula on gastric function and fat digestion in the premature[END_REF][START_REF] Omari | Multipoint measurement of intragastric pH in healthy preterm infants[END_REF][START_REF] Lopez-Alonso | Twenty-fourhour oesophageal impedance-pH monitoring in healthy preterm neonates: rate and characteristics of acid, weakly acidic and weakly alkaline gastroesophageal reflux[END_REF][START_REF] Dipalma | Lipase and pepsin activity in the gastric mucosa of infants, children, and adults[END_REF][START_REF] Henderson | Bioactive Components of Human Milk -Gastric proteolysis in preterm infants fed mother's milk or formula[END_REF]. Therefore the present model must be considered as a screening technique allowing the comparison of different samples digested under the same conditions and that has been applied to purified proteins with limited buffering capacities. Dynamic models integrating pH variations in the stomach and peristalsis would give a clearer idea of what really happens in vivo and will be developed in the future. Furthermore, this study has been conducted on native proteins and it is clear that in "real food", proteins will be submitted to processing conditions (heat-treatment, acidification, hydrolysis, etc) that will significantly affect their structure and consequently their resistance to digestion. All these considerations have been taken into account in a joint project between INRA and IFR that is currently under publication. The next step will then consist in validating this model against data collected in vivo. For ethical reasons, validation towards human won't be possible to carry out.

Figure captions

  Molecular weights markers comprised the following mix of proteins (Invitrogen Carlsbad, CA, USA): insulin -chain (Mr 2,500 Da), insulin -chain (Mr 3,500 Da), aprotinin (Mr 6,000 Da), α-lactalbumin (Mr 14,200 Da), lysozyme (Mr 14,400 Da), trypsin inhibitor (Mr 20,000 Da), carbonic anhydrase (Mr 31,000 Da), lactate dehydrogenase (Mr 36,500 Da), ovalbumin (Mr 45,000 Da), glutamic dehydrogenase (Mr 55,400 Da), bovine serum albumin (Mr 66,000 Da), bovine serum albumin (Mr 66,300 Da), phosphorylase (Mr 97,400 Da) -galactosidase (Mr 116,300 Da), myosin (Mr 200,000 Da).

  digestion. Digests were subjected to nanoscale reverse phase liquid chromatography on a modular LC Packings Ultimate HPLC system equipped with a Famos autosampler and a Switchos microcolumn switching device (LC Packings, Dionex Co., Amsterdam, The Netherlands). Samples were previously concentrated and desalted on a 5 mm x 300 m PepMap C18 precolumn (100 Å, 5 m, LC Packings). The nanoscale LC eluent from the analytical column was sent to the nanoelectrospray ionization source of a QSTARXL global hybrid quadrupole/time-offlight mass spectrometer (Applied Biosystem, Framingham, CA, USA) operated in positive ion mode. Raw data were automatically analysed on a local server harbouring Mascot (Matrix Science, London, UK) and updated Swissprot and NCBI-NR library databanks. Results were corroborated with ProID software (Applied Biosystem, Framingham, CA, USA). Peptides were considered unambiguously identified when their Mascot score indicated significant identity or extensive homology (p<0.05).

  ; Fig. 2b). Digestion products of Mr ~ 22500, 19000 and 12800 Da, appearing after 1 min digestion, showed more resistance, disappearing after 40 min gastric digestion and remaining detectable throughout the gastric stage of digestion in the infant model, indicating that -CN was more rapidly cleaved by the adult model. Polypeptides of Mr ~ 11200, 5700 and 4400 Da were observed after 1-2 min digestion in the adult model, all being degraded by 20 min. In contrast, intermediate Mr ~ 7400 Da and a Mr ~ 4400 Da polypeptides were present in the infant model of digestion, persisting for up to 60 min. OVA showed an intermediate behaviour, being resistant to pepsinolysis in both models (Fig. 1 e, f) but was more rapidly hydrolysed in the adult than in the infant one, with percentages of intact OVA remaining after the gastric stage of 22.3% and 41.1% respectively (Fig. 2c). As for -Lg, the kinetics of appearance of digestion products were different between the two models: while the Mr ~ 21700 and 17800 Da polypeptides were present in the adult model, the latter disappearing after 60 min digestion, an intermediate Mr ~ 20600 Da polypeptide was observed in the infant model, increasing in intensity towards the end of digestion. Lower Mr ~ 5400 and 4600 Da polypeptides were present both in the adult and in the infant model, persisting until the end of digestion with the exception of the Mr ~ 4600 Da polypeptide, which disappeared after 40 min in the adult model. During the duodenal phase, the percentage of intact OVA decreased further with the adult model whereas it remained constant with the infant one (Fig. 1 g, h). Prominent intermediate digestion products of Mr ~ 20700, 17000, 12500, 10400 and 6500 Da were observed during the early stages of adult digestion (1-2 min) and persisted after 30 min. In the infant model only the intermediate digestion products of Mr ~ 20700, 5700 and 5000 Da were present, all of which remained until the end of digestion.

  Immunoreactive digestion products obtained with the adult model, showed several bands of different intensities running around Mr 36, 21, 18, 13, and 6kDa. The pattern of products obtained with the infant model was slightly different, lacking the Mr ~ 21 kDa polypeptides with the other polypeptides being less abundant in the adult model as indicated by their weak staining. Immunoreactivity of -CN was extensively reduced by digestion using both the adult and infant models. No polypeptide digestion products were detected by the anti--CN antiserum in adult digest whereas two faint bands immunoreactive polypeptides were visible around Mr 3 kDa in the infant digest. Finally, intact OVA showed a strong immunoreactive band around Mr 42 kDa which was accompanied by a band running around Mr 80 kDa which probably corresponds to OVA dimers. Following digestion using the adult model, immunoreactive polypeptides were observed around Mr 42, 40, 24, 20, 18, 14 and 10 kDa, many of which were observed in infant model digests but were much more weakly stained.

Figure 1 .

 1 Figure 1. SDS-PAGE analysis of -Lg subjected to duodenal proteolysis using

Figure 2 .

 2 Figure 2. Digestion of -Lg (a), -CN (b) and OVA (c) using an infant (□) or an

Figure 3 .

 3 Figure 3. Evolution of -Lg (a), -CN (b) and OVA (c) residual

Figure 4 .

 4 Figure 4. Western-blotting of (from the left to the right) β-Lg, β-CN and OVA

Figure 5 .Figure 6 .

 56 Figure 5. Residual immunoreactivity of β-CN after the gastric phase of the

Figure 7 .

 7 Figure 7. Peptides identified by LC-MS-MS at the end of β-Lg (a), β-CN (b)

Figure 8 .Table 2 .

 82 Figure 8. β-CN hydrophobicity profile as calculated at pH 3.0 according to

Table 3 .

 3 List of trypsin and chymotrypsin theoretical cleavage sites on ovalbumin and the observation of their hydrolysis () by the adult or infant model.

	Protein Protease Cleavage sites Adult Infant
	OVA	Trypsin
		K16-E17
		K19-V20
		K46-D47	
		R50-T51		
		K55-V56
		R58-F59
		K61-L62
		R84-D85
		K92-P93
		R104-L105
		R110-Y111
		K122-E123
		R126-G127
		R142-E143
		R158-N159
		K181-G182
		K186-A187
		K189-D190
		R199-V200
		K206-P207
		R218-V219
		K226-M227	
		K228-I229
		K263-L264
		R276-K277
		K277-I278	
		K279-V280
		R284-M285
		K286-M287
		K290-Y291
		K322-I323
		R339-E340
		R359-A360
		K369-H370
		R386-C387

Therefore, use of animal models like piglets or young rats will be the only alternative.

Such work is currently in progress.

This work was supported by the