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From computational neuroscience to computational learning science: modeling the brain of the learner and the context of the learning activity

Margarida Romero, Frédéric Alexandre, Lisa Roux, Gérard Giraudon, Thierry

Viéville

To cite this version:

Margarida Romero, Frédéric Alexandre, Lisa Roux, Gérard Giraudon, Thierry Viéville. From computational neuroscience to computational learning science: modeling the brain of the learner and the context of the learning activity. SophIA-summit 2020, Nov 2020, Sophia-Antipolis / Virtual, France. 2020. hal-03018617

From computational neuroscience to computational learning science:

modeling the brain of the learner and the context of the learning activity.

Margarida Romero 1 , Frédéric Alexandre 2 , Lisa Roux 1 , Gérard Giraudon 3 , Thierry Viéville Ill-defined problem engaging the participants in Considering this effective functional model of brain functions allows us to provide an operational model of the learner behavior, in link with machine learning algorithms, such as reinforcement learning.

From wikipedia

a

  task which goal requires to explore the four unknown cubes placed in front of the participant. The affordances are important in the process of understanding the material and its features to configure it in a way to solve the problem (goal state)Goal : "build a vehicle moving from a red point to a black point" In Computational neuroscience and bioinspired artificial intelligence (AI), different studies aim at understanding the mechanisms of perception such as shape recognition and sensori-motor coordination such as grasping or pinching. A step further, as in the Mnemosyne research program, cognitive mechanisms are analyzed to better understand brain circuits responsible for reasoning and problem-solving, by a biological or algorithmic agent. even on simple tasks of problem-solving, we do not yet have a model that encounters the underlying cognitive processes of the learner's brain, in link with the dynamic state of the activity system assessment that takes place at the task level. ⇒ the AIDE research program aims to build such models to better understand the interaction between brain processes and problem-solving activity from a behavioral perspective analysed based on learning analytics generated automatically, also through the human analysis of this activity based on coding schemes. or tangible playful activities with low-cost devices in order to use them in concrete learning environment:Here computational thinking competences related to algorithm, data representation and information coding is targeted, in an autonomous learning setup. Monitoring such activity influences the learning paradigm, involving the learner in a learning to learn process. The French National Direction of Digital Education (DNE) at the Ministry Of Education (MENJS) has selected the LINE research team for being responsible of one of the 9 thematic workforces. The GTnum 9 is devoted to the advancement of Artificial Intelligence in Education. COGNITIVE NEUROSCIENCE MODELING of executive functions allows to model the learner involved in a precise learning task. For instance, the prefrontal cortex encodes the outcome, or value, of an action (orbitofrontal), while action decision making and working memory is located in the dorsolateral part, in link with the basal ganglia for action selection and action reward processing and other brain Structures.


Pauline Rovillain
email: pauline.rovillain@insp.upmc.fr

Ronei Cardoso De Oliveira

Massimiliano Marangolo

Jean-Yves Duquesne

Non-symmetric spin-pumping in a multiferroic heterostructure

We present spin pumping experiments in Co/Pt bilayers grown on Al2O3(0001) and on ferroelectric Y-cut LiNbO3 substrates. Spin pumping is triggered by resonant ferromagnetic resonance induced by surface acoustic waves. We observe that spin pumping efficiency varies when both the magnetization vector and the acoustic wave vector are reversed in Pt/Co/LiNbO3. This phenomenon is not observed in Pt/Co/Al2O3. We propose that the in-plane electric polarization of LiNbO3 is at the origin of the observed phenomenon. These observations open up the perspective of an electric field control of spin pumping efficiency.

I. INTRODUCTION

The first major success in spintronics research was the development of the giant magneto-resistance spin valve, demonstrating the ability to control a spin-polarized current by taking advantage of the spontaneous imbalance of spin-up and spin-down electrons in the conduction band of ferromagnetic materials [1][2][3] . More recently, pure spin currents, i.e. a spin flux without a corresponding charge flux, are of great interest to the scientific community since they do not give rise to an Oersted field and more importantly, they do not produce Joule heating. Pure-spin current devices are therefore a route to reducing the power consumption of spintronic devices such as, for example, in STT-MRAM [START_REF] Maekawa | Spin Current[END_REF][START_REF] Berger | [END_REF][6] .

The development of low-power spintronic devices based on manipulation of pure spin currents requires an increase in the generation and detection efficiencies via a full understanding of the complex physics behind pure spin currents. Spin current can be detected by the inverse spin Hall effect (ISHE) which corresponds to the conversion of a spin current into a detectable charge current by measuring the voltage on a normal metal (NM) presenting a strong spin-orbit interaction (e.g. Pt). Concerning the generation of pure spin currents different techniques have been proposed in the literature. Emission can be obtained by temperature gradients through the spin Seebeck effect [7][8][9] , by charge currents (spin Hall effect 10 ) and by magnetization dynamics (spin pumping 9,11,12 ). This last mechanism permits the generation of a pure spin current via ferromagnetic resonance (FMR) in a ferromagnetic material (FM), leading to spin accumulation in an adjacent non magnetic material.

Generally, FMR is obtained by radio-frequency (RF) electromagnetic means or by RF currents 13 . Interestingly, FMR and spin pumping has also been triggered by acoustic means taking advantage of resonant magnetoelastic coupling in a Co/Pt bilayer 11 . Here, mature surface acoustic wave (SAW) technology is used to drive the dynamics of magnetization of a thin Co film, in the GHz regime and by a remote, non inductive and dissipationless method. This so-called SAW-FMR is now well established [14][15][16] . The precession pumps a spin current into the NM layer 17 . In turn, this spin current generates an electric field (E ISHE ) in the NM layer through the ISHE: E ISHE ∼ J S × σ where J S is the spin-current density and σ correspond to the spin polarization vector. This electric field is converted into a measurable voltage V I between both ends of the Pt strip. What makes SAW assisted experiments extremely interesting is that they are free from spurious non-symmetric signal arising from the anomalous Hall effect (AHE) which affects the standard FMR technique 18 .

The main purpose of this letter is to compare the spin pumping excitation/detection efficiency when both directions of the magnetization M and acoustic wave-vector k SAW are reversed. We investigated SAW-FMR assisted spin pumping in a multiferroic system where a FM/NM (Co/Pt) bilayer is in contact with a ferroelectric (FE) LiNbO 3 substrate. We put forward the hypothesis that the remanent electrical polarization vector affects the ISHE voltage.

II. SAMPLES CHARACTERISTICS AND EXPERIMENTAL SETUP

Figure 1(a) is a sketch of the samples and setup. We used two different substrates: Y-cut LiNbO 3 and (0001)cut Al 2 O 3 . Moreover, LiNbO 3 is not centrosymmetric (space group is R3m). It is ferroelectric and therefore piezoelectric. We verified that our LiNbO 3 substrate is a single-domain ferroelectric using polarized light microscopy and piezoelectric force microscopy (PFM) measurements (see appendix A, Fig. 4 and5). No domain wall was observed from large scale (2.86 mm × 2.11 mm) to low scale (1 µm × 1 µm) that is expected in a singledomain ferroelectric material. Al 2 O 3 is centrosymmetric (space group is R3m). It is neither piezoelectric nor ferroelectric. The orientations of both substrates have been verified by XRD. Both samples do not present a 2-fold rotational symmetry about the surface normal. On both substrates, a 500µm × 1.5mm bi-layer of Co (10nm) and Pt (7nm) is deposited by electron beam evaporation at the center. SAW bursts are emitted and detected electrically by means of two nominally identical IDTs defined by electron beam lithography and placed symmetrically on either side of the central Co/Pt strip. In the case of the Pt/Co/LiNbO 3 sample, IDTs are deposited directly onto the piezoelectric substrate. However, in case of the Pt/Co/Al 2 O 3 sample, a thin ZnO piezoelectric layer of 490 nm is deposited on the surface by sputtering at 300 • C only under the IDTs areas so as to emit SAWs. ZnO is a textured polycrystal with c-axis normal to the substrate.

200ns SAW bursts, with a repetition rate of 5 kHz, are emitted at 1.5 and 1.3 GHz for LiNbO 3 and Al 2 O 3 respectively. The electrical power applied during the SAW bursts was 19 dBm (79 mW), except for power study in Fig 2. After propagation in the Co/Pt layers the SAW is detected by the opposite IDT and the acoustic signal is acquired with a digital oscilloscope. Simultaneously, the output voltage V (t) (between the both ends of the Co/Pt bi-layer) is measured with a DC-amplifier (bandwidth = 0-20 MHz, gain = 25 dB, input and output impedance = 50 Ω) and recorded with the same oscilloscope. To improve the signal-to-noise ratio, a sequence of 64000 V (t) signals is averaged.

On the Y-cut LiNbO 3 substrate, the SAW wave-vector k SAW is along the Z-axis ([0001] direction), i.e. the spontaneous direction of the electrical polarization vector P 19,20 . On the Al 2 O 3 substrate, k SAW is along the Y-axis ([1 100] direction). On both substrates we define x parallel to k SAW , oriented from IDT 1 to IDT 2 . We also define an in-plane axis x at an angle φ from x. We apply a magnetic field B ext along x (see Fig. 1(a)).

The DC spin current is detected via the ISHE in the Pt layer by measuring the time-dependent voltage between both ends of the Co/Pt bi-layer. The output voltage V (t) is recorded synchronously with the acoustic bursts (see Fig. 1(d,e)). To separate, in the time domain, the spin current signal V from the electromagnetic wave (EMW) and from the acoustic (SAW) signals, the distances between each IDT and the bi-layer is carefully designed (1.25 and 2.75 mm for LiNbO 3 and Al 2 O 3 , respectively). In an experimental run, we first set the orientation φ and we saturate the magnetic film with a large field amplitude B sat . Then, the field either increases (from -35 to +35 mT, B sat = -350 mT) or decreases (from +35 to -35 mT, B sat = +350 mT). V (t) is recorded at each field value. These measurements were performed for a range of orientations φ and also for opposite acoustic wave-vectors k SAW . The direction of k SAW is reversed by connecting the input signal to one IDT or to the other. Since the IDTs are symmetrical with respect to the Co/Pt strip, spin pumping occurs in the same time range [t 1 , t 3 ] for both propagation directions.

For each sample measured, the Co layer was first characterised since each evaporated layer has slightly different characteristics (see appendix B). In this paper, we choose to present the results obtained for two specific samples where the coercive field B c is lower than the resonant field B res .

From broadband-FMR (BB-FMR) measurements between 3 and 8.5 GHz, we record the FMR frequency, F FMR , as a function of the in-plane magnetic field amplitude (B ext ) 21 . By extrapolation of our experimental values (F FMR vs B ext ) using the Kittel formula 22 , we found a SAW-FMR resonance condition around 4 mT and 6 mT for Pt/Co on LiNbO 3 and Al 2 O 3 , respectively. Both samples are invariant by a rotation of π, regarding their MOKE magnetic cycles (see appendix B and insert of Fig. 6). First, we present our results. Afterward, we will analyse them in term of symmetries.

III. RESULTS AND DISCUSSION

Measurements, presented here, have been performed at φ = 0 • with B c = 1.1 mT for Pt/Co/LiNbO 3 and φ = 60

• with B c = 3 mT for Pt/Co/Al 2 O 3 . Figure 1(e) shows the spin current signal ∆V (t) = V (t, B ext ) -V (t, B ref ) for Pt/Co/Al 2 O 3 , B ext = ±5 mT and B ref = ±35 mT.
This subtraction is performed to eliminate parasitic deterministic signals. According to calculation, no SAW-FMR and no spin pumping are expected at ±35 mT. On the contrary, at B ext = ±5 mT, the acoustic frequency matches the precession frequency of the uniform mode (See appendix B, Fig. 8). Hence, when the acoustic bursts go through the Co/Pt bi-layer, in the time range [t 1 ,t 3 ] ([0.6-0.9] µs in the case of the Pt/Co/Al 2 O 3 sample, Fig. 1 (e)), they induce the ferromagnetic resonance of Co and a pure spin current is launched in the adjacent Pt layer, with a maximum value around t 2 . The magnitude of the spin current is proportional to the timeaveraged value V I of ∆V (t), around t 2 (between 0.7 and 0.8 µs for Pt/Co/Al 2 O 3 ).

We assume that the spin current is maximum when the resonance is triggered. Then, we identify the resonance field B res at the value where the current signal V I is maximum (see Fig. 3). We observe the change of sign of V I with the reversal of M, induced by the reversal of B ext (since B c < B res ), which is an expected condition for acoustic spin pumping 23 . We also note that V I (proportional to the spin current) is proportional to the incident electrical power (P el ), and then to the electrical power injected in the IDT, as shown in Fig. 2 for the bilayer on the Al 2 O 3 substrate. The same behavior is observed for the LiNbO 3 sample. Indeed, this is in good agreement with experiments of K.Ando et al. 12 and with the model of F.Czeschka et al. 23 , from which we can easily derive, in the small angle regime: V I ∝ P el .

At first glance (see Fig. 3), the V I dependence on B ext shown here seems to be similar to that shown by M.Weiler et al 11 . However, a difference is observed in our measurements at the coercive fields. Indeed, a significant electric current is emitted at -B c and +B c for decreasing and increasing field respectively, which is difficult to observe in M.Weiler et al 11 . The origin of this current is not yet understood and is beyond the scope of this paper. However, since B c is close to the resonant field, the current superimposes on the spin current and it becomes difficult to separate the two contributions. To avoid the spurious contributions, we concentrate only on the measurements of V I for low coercive field samples and at angles φ where B c is weakest.

So, let us now see what is expected when the magnetic field and the acoustic wave-vector are reversed. Figures 1(b,c) displays sketches of a sample (substrate and bilayer) subject to two configurations (b) and (c), i.e. different orientations of in-plane applied field B ext , magnetization M and acoustic wave-vector k SAW . Configuration (c) is obtained from (b) by reversing B ext , M and k SAW . A trivial behaviour is expected for systems that are invariant by a rotation of π about an axis perpendicular to the propagation plane. In that case, we can notice that configuration (c) can also be obtained from (b) by combining a rotation of π and an exchange of the (+) and (-) terminals. Therefore, if the sample is invariant by a rotation of π, we infer that going from (b) to (c) will change the sign of the measured voltage between the (+) and (-) terminals but not its magnitude.

From an experimental point of view, reversing k SAW and B ext is quite easy. However, reversing M is a little bit more tricky because of the memory effect associated with the magnetic hysteresis. In Fig. 1, if configuration (b) is reached from a positive saturating field ("decreasing field scan"), configuration (c) must be reached from a negative saturating field ("increasing field scan"), and conversely. To B dec ] scans) do not superimpose. For example, at B ext = 5 mT, the magnitude of the voltage is either 2.5 or 5 µV, depending on the scans. This factor of 2 cannot be attributed to possible different intensities of the acoustic field, at k SAW and -k SAW , arising from different efficiencies of the IDT 1 and IDT 2 transducers. If a multiplying factor were applied to correct the amplitudes at 5 mT, then the difference in amplitudes at -5 mT would further increase. We observed this for all angles φ and samples studied even if B c > B res . This phenomenon must not be confused with "non-reciprocal" acoustic propagation observed in magnetic materials where only k SAW is inverted 25 .

Fig. 3 (c,d) displays our results for the Pt/Co/Al 2 O 3 sample. The curves obtained for corresponding field scans superimpose. Reversing simultaneously k SAW , B ext and M changes the sign of the measured voltage but not its magnitude. Indeed, slightly different amplitudes are observed which can be attributed to different intensities of the acoustic field, at k SAW and -k SAW , arising from different efficiencies of the two transducers. The same multiplying factor of 1.1 can be applied to correct the amplitudes at 5 mT and at -5 mT.

In the Pt/Co/LiNbO 3 sample, the origin of the effect has to be determined. Fundamentally, the non-symmetry of the current originates from the non-invariance of the system by a rotation of π (about an axis perpendicular to the propagation plane). A tentative explanation involves the polarization vector, P, that is oriented along the inplane Z direction. One possibility concerns the excitation process and involves a coupling between the ferroelectric polarization P of LiNbO 3 and the ferromagnetic moment of Co, as seen in magnetoelectric materials 26 or in artificial composite multiferroic systems [27][28][29] where the P vector is oriented perpendicularly to the FE/FM interface, permitting an efficient coupling of ferroic orders. We point out that in Y-cut LiNbO 3 substrates, P lies parallel to the interface. In that configuration, the electrical field in the cobalt thin film goes to zero. Nevertheless, we put forward the hypothesis that the interface roughness can induce interface charge accumulation and consequent non equilibrium spin density along the out-ofplane direction. As pointed out in Jia et al. 28 , this spinunbalanced interface electrostatic effect accompanied by the so-called s-d exchange interaction, may give rise to an effective magneto-electric (ME) coupling affecting the whole Co thin film and the consequent spin pumping efficiency at the Co/Pt interface. This could modify either the magnetic precession cone angle at resonance, or the magnetic precession frequency, or both, and then modify the spin current generation. Nevertheless the BB-FMR measurements that we performed did not show any evidence of an FMR resonance shift after magnetic field reversal. This hypothesis can be tested in the future by a systematic study of non-symmetric spin pumping as a function of the interface roughness and/or the misalignment of the normal to the surface. To compare with the experiments performed on Pt/Co/Al 2 O 3 (which display invariance by a π-rotation), we notice that the Al 2 O 3 structure is also not invariant by a π-rotation since the symmetry about (0001) is only of order 3. However, a 2π/6 rotation leaves unchanged the location of the oxygen atoms and only involves a change in the orientation of the aluminium triangles which are located between the oxygen planes. Therefore, in our Pt/Co/Al 2 O 3 sample, the non-invariance by a rotation of π is somehow "weak" since it is just due to slight variations of atoms stacking.

Other processes involving the detection can be evoked due to the electrical field in the Pt layer, arising from P. However, they must be very weak, because of the geometry and of screening effects. In order to corroborate our hypothesis it would be interesting to quantify the effect of electrical polarization on spin current generation by modifying or reversing P through the application of an electric field. Unfortunately, the fields required to reverse the polarization are beyond our technical capabilities. Indeed, the coercive electric field of the LiNbO 3 is E c > 210 kV.cm -130 which would require to apply on our samples a voltage U > 21 kV.

IV. CONCLUSION

In conclusion, we have investigated the acoustic spin pumping in Co/Pt bilayers deposited on LiNbO rotational symmetry of spin-pumping signal that we possibly attribute to the permanent electrical polarization of LiNbO 3 . We suggest that this polarization is an important factor in the spin pumping excitation processes. The nature of the coupling with the magnetic polarization is still unclear. It may be due to a magnetoelectric coupling at the interface between LiNbO 3 and Co, inducing non equilibrium spin density across the whole thin film 28 and affecting spin pumping efficiency. It will be interesting to use a ferroelectric substrate whose polarisation P can be easily varied, in order to modify the spin current emission in a bilayer.

Appendix A: Single domain evidence in ferroelectric LiNbO3

To verify that the LiNbO 3 substrate is a single ferroelectric domain, we observed a substrate (from a series) using polarized light microscopy. Fig. 4 summarizes our observation on a large scale (2.86 mm × 2.11 mm) for a range of angles, in a rotation around the normal to the substrate. As expected, the intensity of the transmitted light depends on the angle. However, no contrast is observed over the sample, suggesting a single domain configuration. Nevertheless, anti-parallel domains cannot be excluded. To clear this point, we observed the sample (using polarized light microscopy) on a much smaller scale (110 µm × 83 µm) in an attempt to reveal walls between anti-parallel domains. According to V.G.Zalessky et al. 31 , domain walls induce optical contrast on this scale. Moreover, piezoelectric force microscopy (PFM) measurement is performed in order to detect micro-and/or nano-metric ferroelectric domains. Figures 5 (a,b,c) show PFM images for a large scale (20 µm × 20 µm) and (d,e,f) show PFM images for a smallest scale (1 µm × 1 µm). It turns out that ou-of-plane and in-plane measurements do not put into evidence any ferroelectric domain for both scale. We observe only a line which is present as well in the topographic and piezoelectric mode indicating that this line is a defect on the surface. Since we observed neither walls and domains, we conclude that our LiNbO 3 substrates exhibit a single ferroelectric domain configuration 31,32 . and -M (-B)/M S , respectively. The nearly perfect superposition indicates that the magnetic layer is invariant by a rotation of π (same for Co on Al 2 O 3 , not shown).

The magnetic cycle is recorded for different angles.

FIG. 1 .

 1 FIG. 1. (a) Sketch of the sample and setup. On the LiNbO3 and Al2O3 samples, IDTs have 30 pairs of fingers with an aperture of 500 µm. They are made of Al with a metallization ratio equal to 0.5. On the LiNbO3 sample, the distance between IDTs is 3 mm and between one IDT and the Co/Pt bilayer is 1.25 mm. On the Al2O3 sample, the distance between IDTs is 6 mm and between one IDT and the Co/Pt bilayer is 2.75 mm. (b,c) display the sample, with two configurations of in-plane external magnetic field Bext, magnetization M and k SAW . (c) is obtained from (b) by inversion of Bext, M and k SAW . Spin current signal V is measured between the (+) and (-) terminals. (d) Output of the receiving IDT as a function of time. EMW signal and SAW pulses are observed. (e) ∆V (t, Bext) = V (t, Bext) -V (t, B ref ) where V is the spin current signal. Bext = ±5 mT. B ref = ±35 mT (see main text). Pt/Co/Al2O3 sample.

FIG. 2 .

 2 FIG. 2. VI (proportional to the spin current amplitude) as function of incident electrical power on IDT for Pt/Co/Al2O3 sample 24 . The red line is a linear fit.

  conclude, in order to check the symmetry rules, we should compare the voltages measured either for [k SAW , B ext , B dec ] and [-k SAW , -B ext , B inc ], or for [k SAW , B ext , B inc ] and [-k SAW , -B ext , B dec ]. From a practical point of view, the comparison can be easily carried out in the following manner. Let us consider two field scans, [k SAW , B inc ] and [-k SAW , B dec ]. For the first scan, we plot the voltage V I versus the signed amplitude of the field. For the second scan, we plot the opposite voltage -V I versus the opposite of the signed amplitude of the field. Fig.3 (a,b) displays our results for the Pt/Co/LiNbO 3 sample. Clearly, the curves obtained for [k SAW , B dec ] and [-k SAW , B inc ] scans (or [k SAW , B inc ] and [-k SAW ,

FIG. 3 .

 3 FIG. 3. (a, b) Co/Pt on LiNbO3 and (c, d) Co/Pt on Al2O3. VI (proportional to the spin current) versus Bext after symmetry operations on -k SAW scans. Black and blue curves: raw data. Orange and green curves: starting from raw data, VI and Bext are multiplied by -1 (see main text). (a, c) Comparison between [k SAW , B dec ] and [-k SAW , Binc]. (b, d) Comparison between [k SAW , Binc] and [-k SAW , B dec ].

  3 and Al 2 O 3 substrates. Our experiments pinpoint the lack of π

FIG. 4 .

 4 FIG. 4. Cross-polarized light intensity transmitted through a LiNbO3 substrate, as a function of the angle of the substrate. Inset images: pictures under cross polarized light microscopy (2.86 mm × 2.11 mm). Numbers link images and intensities, at a given angle.

FIG. 5 .

 5 FIG. 5. PFM images of a surface of LiNbO3 substrate. (a,b,c): 20 µm × 20 µm image size, (d,e,f): 1 µm × 1 µm image size. (a,d) correspond to topography measurements of the surface. (b,e) show the response of out of plane piezoelectricity and (c,f) the in plane piezoelectricity one.

FIG. 6 .

 6 FIG. 6. Magnetic cycles M (B)/MS for Pt/Co/LiNbO3 (at φ = 0 • ) and Pt/Co/Al2O3 (at φ = 60 • ) samples. Insert: Pt/Co/LiNbO3 (at φ = 0 • ). Comparison of M (B)/MS and -M (-B)/MS.

Figure 7 displays

 7 B c as function of the angle, we can see that the magnetic properties are different between the two Co layers as the easy axis and the magnitude of B c do not match.

Figure 8

 8 Figure 8 represents the FMR frequency, F FMR , as a function of the in-plane magnetic field amplitude (B ext ), from broadband-FMR (BB-FMR) measurements between 2.5 and 8.5 GHz. By extrapolation of our experimental values (F FMR vs B ext ) using the Kittel formula 22 on both Co/Pt samples, we found a SAW-FMR resonance

FIG. 8 .

 8 FIG. 8. (black square) Experimental FMR frequencies of the Co layer on LiNbO3 as function of the external magnetic field. (black curve) Extrapolation by the Kittel formula. (Magenta curve) SAW excitation frequency.

ACKNOWLEDGMENTS

The authors acknowledge L.Becerra and M.Rosticher for optical and electronic lithography, S.Suffit for e-beam evaporation of Co/Pt bilayers, F.Vidal for assistance during MOKE experiments, Y.J. Zheng for X-ray diffraction measurements, H.Cruguel for PFM measurements and C.Gourdon, L.Thevenard and P.Atkinson for careful reading.

Appendix B: Magnetic properties

From the magnetic point of view, each evaporated Co layer has different characteristics. Figure 6


Céline

Elissa Abi-Habib

Céline Poncet-Legrand

Stéphanie Roi

Stéphanie Carrillo

Thierry Doco

Aude Vernhet
email: aude.vernhet@supagro.fr

Impact of grape variety, berry maturity and size on the extractability of skin polyphenols during model wine-like maceration experiments

Keywords: grape skins, alcohol insoluble solids, extraction, anthocyanins, tannins, chemical reactivity

Background

Skin cell walls modulate anthocyanin and tannin extraction from grape skins. However, relationships between the composition of alcohol insoluble cell wall solids (AIS) and extraction are still unclear.

Our objectives were to characterize the impact of variety, berry size and ripeness on skin AIS composition (polysaccharides, proteins) and polyphenol extraction during maceration. Results. The grape skin composition and its impact on polyphenol extraction was compared for two varieties, Carignan and Grenache, with skins of berries sorted according to their size and density. Extractions were performed in model wine-like maceration conditions. Fresh skins had similar contents in polymeric tannins, but strongly differed by their anthocyanin contents (higher in Carignan and in the ripest berries) and composition (higher proportions in coumaroylated anthocyanins in Carignan).

Anthocyanin extraction was proportionally much higher in Grenache, which was not just related to the Carignan's higher levels in coumaroylated anthocyanins. Chemical reactions decreased anthocyanin concentrations in solution for both varieties. Tannin extraction for Grenache was slightly higher and faster than for Carignan. Skin AISs differed slightly between the two varieties by their carbohydrate composition and protein content, but not between modalities. Polyphenol analyses in the precipitates evidenced at the end of the maceration and in residual skins highlighted differences between the two varieties and between berries with different ripeness.

Conclusion.

Structural information on the cell wall network and on its changes during maceration, along with a better understanding of the chemical reactions of anthocyanins and tannins is needed to better relate grape and wine polyphenol composition.

INTRODUCTION

Anthocyanins and condensed tannins (flavan-3-ol polymers, also referred to as proanthocyanidins) are responsible for the colour and mouthfeel of red wines and play an important part in their quality. Anthocyanins and tannins in skin cells are mostly located in vacuoles, tannins being also found associated with the cell walls [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF][START_REF] Joutei | Localization of tannins in grape berry skins[END_REF] . Tannins are also extracted from seeds, although in lower extent than from skins. Tannin and anthocyanin extraction from the skins/seeds of grapes during winemaking is not total and several studies have shown that there is no direct relationship between their contents in grapes from different varieties and those found in the corresponding wines [START_REF] Hazak | The phenolic components of grape berries in relation to wine composition[END_REF][START_REF] Bautista-Ortín | The Extraction Kinetics of Anthocyanins and Proanthocyanidins From Grape To Wine[END_REF] . The latter depend on the extraction conditions (ethanol content, temperature, maceration length, …), on the berry size (skin surface to juice ratio [START_REF] Singleton | Effects on red wine quality of removing juice before fermentation to simulate variation in berry size[END_REF] and possibly skin composition [START_REF] Wong | Combined physiological, transcriptome, and cis-regulatory element analyses indicate that key aspects of ripening, metabolism, and transcriptional program in grapes (Vitis vinifera L.) are differentially modulated accordingly to fruit size[END_REF] ), and on the cap-management practices [START_REF] Boulton | Principles and practices of winemaking[END_REF][START_REF] Setford | Factors affecting extraction and evolution of phenolic compounds during red wine maceration and the role of process modelling[END_REF] during winemaking, as well as on their ability to cross the barriers represented by cell structure and in particular cell walls [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF][START_REF] Hernández-Hierro | Relationship between skin cell wall composition and anthocyanin extractability of Vitis vinifera L. cv. Tempranillo at different grape ripeness degree[END_REF] . Other factors that modulate anthocyanin and tannin concentrations in wines are: (i) their adsorption on insoluble pulp debris (flesh cell walls) [START_REF] Bindon | Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass[END_REF] and on yeasts 11 ; (ii) their interactions with soluble grape compounds (polysaccharides, proteins) extracted during maceration, leading to precipitations [START_REF] Bindon | Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components[END_REF] ; iii) their chemical reactivity. Indeed, once extracted, anthocyanins and tannins undergo several chemical reactions that profoundly change their composition throughout the process [START_REF] Cheynier | Structure and properties of wine pigments and tannins[END_REF][START_REF] Hanlin | Review: Condensed tannin and grape cell wall interactions and their impact on tannin extractability into wine[END_REF] . Not all the compounds formed can be identified and quantified, which may contribute to the lack of relationship observed between the polyphenol composition of grapes and that of red wines at the end of the maceration. This relationship, related to several factors, is difficult to model, even under constant process conditions and even though this knowledge would be of great interest for the selection of new grape varieties or for the adaptation of winemaking techniques to different raw materials. Experiments under model conditions, associated with the characterisation of grapes and wines are therefore necessary in order to compare the varieties, dissociate the various phenomena and highlight those factors likely to play a predominant role in the polyphenolic composition of wines.

When dealing with polyphenol extraction from skins, cell walls are considered as one of the main factor that control their final composition in wines [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF][START_REF] Bindon | Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass[END_REF] . Tannin extraction especially is limited by their interactions with skin insoluble cell wall constituents, i.e. polysaccharides and proteins 15 . Interactions between tannins and cell wall polysaccharides were proven by means of adsorption experiments using purified cell walls and proanthocyanidins [START_REF] Bindon | Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass[END_REF] . Both pectins and hemicelluloses play an important part in these interactions [START_REF] Castro-López | Role of cell wall deconstructing enzymes in the proanthocyanidin-cell wall adsorptiondesorption phenomena[END_REF][START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF] . When in solution, the degree of methylation of pectins increases their interactions with proanthocyanidins [START_REF] Poncet-Legrand | Interactions between Flavan-3-ols and Poly(L-proline) Studied by Isothermal Titration Calorimetry: Effect of the Tannin Structure[END_REF] . Although they are minor components compared to polysaccharides, proteins have a much stronger affinity for tannins [START_REF] Nunan | Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)[END_REF] . They account for about 10% of the insoluble cell wall components and are mainly structural proteins rich in hydroxyproline, proline and glycine [START_REF] Carpita | Structural models of primary cell walls in flowering plants: Consistency of molecular structure with the physical properties of the walls during growth[END_REF][START_REF] Vicens | Changes in polysaccharide and protein composition of cell walls in grape berry skin (Cv. Shiraz) during ripening and over-ripening[END_REF] . Their role in the extractability of tannins, less studied, has been mentioned by several authors [START_REF] Springer | Protein-precipitable tannin in wines from vitis vinifera and interspecific hybrid grapes (Vitis ssp.): differences in concentration, extractability, and cell wall binding[END_REF][START_REF] Ortega-Regules | Anthocyanins and tannins in four grape varieties (Vitis vinifera L.) evolution of their content and extractability[END_REF] . Changes in skin/pulp cell wall composition occur during berry ripening, leading to a loosening of cell walls and fruit softening. Among these changes, a decrease of pectins, related to their solubilization, has been found [START_REF] Vicens | Changes in polysaccharide and protein composition of cell walls in grape berry skin (Cv. Shiraz) during ripening and over-ripening[END_REF][START_REF] Ortega-Regules | Anthocyanins and tannins in four grape varieties (Vitis vinifera L.) evolution of their content and extractability[END_REF] . These structural modifications have been hypothesized to induce changes in skin cell wall structure (porosity, accessibility to interaction sites) and rigidity that may modulate tannin [START_REF] Bindon | Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins[END_REF] or anthocyanin extraction [START_REF] Segade | Berry skin thickness as main texture parameter to predict anthocyanin extractability in winegrapes[END_REF] . It has also been suggested that anthocyanins may influence tannin extraction/solubility and that high anthocyanin/tannin ratios induce higher tannin concentration in wines, regardless of the initial tannin concentration in fruits [START_REF] Kilmister | A role for anthocyanin in determining wine tannin concentration in Shiraz[END_REF] .

The objectives of this work were to: (i) characterize the impact of grape variety, berry size and ripeness on the extraction and evolution of anthocyanins and tannins during skin maceration; (ii) examine links between extraction and skin composition in insoluble materials; iii) identify the main mechanisms involved. Experiments were performed in wine-like model conditions for two contrasted varieties in terms of anthocyanin/tannin ratios (Carignan and Grenache), in the absence of pulp components (to avoid the impact of adsorption/precipitation events not related to skin composition) and fermentation (to avoid chemical changes related to reactions with yeast metabolites and adsorption by yeast cells). Most of the previous studies were performed on varieties harvested through time at different degree of ripeness [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF][START_REF] Hernández-Hierro | Relationship between skin cell wall composition and anthocyanin extractability of Vitis vinifera L. cv. Tempranillo at different grape ripeness degree[END_REF][START_REF] Bindon | Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins[END_REF][START_REF] Fournand | Accumulation and extractability of grape skin tannins and anthocyanins at different advanced physiological stages[END_REF] . Grape heterogeneity in terms of berry size and ripeness was considered here at technological maturity.

MATERIALS AND METHODS

Chemicals

Acetonitrile, methanol, ethanol, acetic acid and formic acid were HPLC grade from VWR. KOH Titrisol 1M was purchased from Merck. Acetone, D (+) galacturonic acid hydrated were provided by Fluka. Sodium chloride, tartaric acid, epicatechin, epigallocatechin gallate, lithium chloride, N,N-dimethylformamide, trifluoroacetic acid, myo-inositol, allose, m-hydroxydiphenyl (MHDP), alcohol oxidase from Pichia pastoris, norleucine, the 18 amino-acid standard kit and hydrochloric acid 37 % were provided by Sigma-Aldrich, sulphuric acid by Roth. Sodium hydroxide 1M was obtained from Fisher. The lithium citrate loading buffer was obtained from Biochrom.

Flavanol dimer B2, flavanol trimer C1 and Malvidin-3-O-glucoside chloride were purchased from Extrasynthese (Genay, France). Ultra-pure water was obtained from a Milli-Q Advantage A10 system (Millipore).

Grape sampling

Two Vitis vinifera grape varieties (Carignan and Grenache) were harvested at an average potential alcohol of 12 % vol. in the vineyard of the Pech Rouge experimental unit (INRAE, Gruissan, France). The berries were sorted according to their natural heterogeneity in terms volume (vol) and density (degree of maturity: deg). This heterogeneity was determined on 1000 berries the day before the harvest by measuring their diameter and estimating their density by flotation in different salt solutions, corresponding to total soluble solid difference between two successive baths of 1 % vol potential alcohol. Berries were recovered by cutting them at the pedicel level and sorted first as a function of their size using a grading machine (vol -, vol + ) and then as a function of their density (deg - , deg + ) using an aqueous solution of concentrated rectified grape must at the adequate density (Fig. 1A). The cut-off for berry sizes and sugar concentration were defined based on the median population of these 1000 berries. The oenological characteristics of the four batches (vol -deg -, vol -deg + , vol + deg - and vol + deg + ) obtained for the Carignan and Grenache varieties are presented in supplementary data S1. Samples of 180 berries of each modality were recovered. Grape skins were separated from the berries with a scalpel and immediately used for diffusion experiments or frozen in liquid nitrogen and stored at -80°C for later analysis of their composition.

Preparation of skin alcohol insoluble cell wall material (AIS)

Frozen skins of each variety and modality (from 30 berries, triplicates) were ground in liquid nitrogen. The alcohol insoluble cell wall solids (AISs) were then isolated from the powders using the procedure described in Apolinar-Valiente et al. [START_REF] Apolinar-Valiente | Application and comparison of four selected procedures for the isolation of cell-wall material from the skin of grapes cv. Monastrell[END_REF] , with slight modifications. AISs were prepared in triplicate and analysed separately. Skin powder (5 g) was suspended in 15 ml boiling water for 5 min and homogenized. One part of the homogenized material was purified with two parts of 96% ethanol for 30 min at 40°C in an ultrasound bath. The alcohol insoluble solids (AIS) were separated by centrifugation and extracted again with 70% ethanol for 30 min at 40 °C. A sample from the liquid phase was taken for soluble sugar assay, done with the sulphuric phenol method. When no more sugar was detected, AISs were further washed twice with 96% ethanol and once with acetone. After being dried with an air flux overnight, they were weighed and used for the following analyses.

Carbohydrates composition of AISs

The neutral sugar composition of the AISs was determined by gas chromatography after polysaccharide hydrolysis with 72% sulphuric acid at 100°C for 3h 29 and conversion of neutral sugars into volatile alditol acetates [START_REF] Harris | An improved procedure for the methylation analysis of oligosaccharides and polysaccharides[END_REF] . Inositol and allose were used as internal standards. The alditol acetates were quantified by gas chromatography with flame ionization detection (GC-FID) (GC 2010 Plus Shimadzu) using a DB225 (30 m × 0.25 mm ID, 0.25 µm film) capillary column and hydrogen 5.6 B50 as the carrier gas. Calibration was done with commercial monosaccharides. Uronic acids were determined colorimetrically in triplicates by the m-hydroxydiphenyl method [START_REF] Blumenkrantz | New method for Quantitative Determination of Uronic Acids[END_REF] . The AISs were first submitted to pre-hydrolyse by the action of sulfuric acid, as described by Ahmed and Labavitch [START_REF] Ahmed | Method for accurate determination of cell wall[END_REF] . A calibration curve was built using pure galacturonic acid solutions (0 to 100 mg/L). The degree of methylesterification of uronic acids (DE) was measured by saponification of the AIS pectins in the presence of KOH, thus allowing the release of methanol. Methanol was converted to formaldehyde that was determined using the colorimetric method of Klavons and Bennet 33 .

Amino acid composition of AISs

Cell wall material (5 mg) was hydrolysed in 1 mL of 6N HCl for 24 h at 120°C. Norleucine was added as an internal standard. After evaporation of the acidic aqueous solution under air stream, samples were washed twice in water and then in 95% ethanol. Finally, samples were dissolved in a 0.2 M pH 2.2 lithium citrate loading buffer and filtered through a 0.22 µm filter (Millipore Millex-GV). Amino acids were quantified by ion exchange chromatography with a Biochrom 30 amino acid analyzer (Biochrom, Cambridge, England), as described in Vicens et al. [START_REF] Vicens | Changes in polysaccharide and protein composition of cell walls in grape berry skin (Cv. Shiraz) during ripening and over-ripening[END_REF] .

Polyphenol extraction from skins and precipitates.

Frozen initial skins and frozen skins after extraction in wine-like conditions were finely ground to a fine powder in liquid nitrogen and using a mortar grinder (Pulverisette 2, Fritsch). 150 mg of powder were treated first with methanol (750 µL) then extracted with 5.25 mL of 60/40/1 (v/v/v) acetone/water/formic acid at room temperature on an orbital shaker (Precellys 24, Bertin technologies, program 5000-3*40-20). The methanolic and acetone/water/ formic acid extracts were pooled and after centrifugation (3000 rpm, 5 min, 4°C), 1mL aliquots were dried in a rotary evaporator under vacuum at 35°C for 2h (EZ-2 plus, Genevac SP service). Dried extracts were redissolved in a wine-like solution (ethanol 12%, 3 g/L tartaric acid, 50 mM NaCl, 40 mg/L SO2, pH 3.5) for UV-visible spectrophotometry and High Performance Liquid Chromatography (HPLC) analysis, or in dimethyl formamide for High Performance Size Exclusion Chromatography (HPSEC). The same procedure except grinding was applied for the extraction and analysis of polyphenols in the precipitates recovered at the end of the wine-like maceration experiments.

Polyphenol analysis.

Total polyphenols Index (TPI) and total red pigments (TRP) were determined by UV-visible spectrophotometry (spectrophotometer UV-1800, Shimadzu) at 280 and 520 nm (1 cm path length) after adequate dilution in HCl 1 M. Free anthocyanins were analysed by HPLC using a Waters chromatography system equipped with DAD detection and a C18 reversed-phase column (Atlantis T3, Waters) as described by Fournand et al. [START_REF] Fournand | Accumulation and extractability of grape skin tannins and anthocyanins at different advanced physiological stages[END_REF] . Anthocyanins were quantified at 520 nm, in equivalent of malvidin-3-O-glucoside.

The size distribution of polyphenols in the samples and the concentrations of polymeric tannins (in eq. epicatechin) were determined by HPSEC, according to the procedure described before [START_REF] Vernhet | Fate of anthocyanins and proanthocyanidins during the alcoholic fermentation of thermovinified red musts by different Saccharomyces cerevisiae strains[END_REF] . Commercial epicatechin, B2 dimer, epigallocatechin gallate, malvidin and home prepared and characterized tannin fractions were used to evaluate retention times corresponding to monomers, oligomers and polymers (supplementary data S2).

Extraction of skin phenolic compounds by diffusion in model wine-like solution

Thirty berries of each modality were manually peeled and fresh skins were weighed and immediately immersed in 42 mL of a model solution containing 3 g/L tartaric acid, 50 mM NaCl and 40 mg/L SO2, at pH 3.5 (adjusted with NaOH 1M). This value (42 mL) was chosen from the data obtained by grape sampling (average weight of berries, skins, seeds, pulp) and microvinifications of 900 g of berries in triplicate of each modality (average volumes of wine obtained for 900 g of berries).

These averages slightly differed depending on the berry size, and the volume varied between 28-31 mL (vol-) and 38-44 mL (vol+). The value of 42 ml corresponded then to the vol+ modalities and the highest liquid to solid ratios, to avoid differences in extraction between modalities related to different solid-liquid equilibria. Simulated maceration experiments were carried out by increasing stepwise the ethanol content from 0 to 15% (Fig. 1B). All experiments were performed in triplicate. Flasks were placed under argon and gently stirred in dark at 22 °C. Polyphenol diffusion was followed during 11 days by measuring the TPI and TRP daily, as well as the HPLC and HPSEC profiles of samples taken and centrifuged (15000 g, 15 min, 15°C) at the end of each ethanol increase step. The dilution induced by the sampling and the addition of ethanol was considered. To account for differences in initial fresh skin mass (dependent on the modality), results were divided by this initial mass and reduced to 1 g fresh skin for all modalities. At the end of the diffusion, skins were recovered, and the diffusion solutions clarified by centrifugation. Skins were then washed first with 0% (3 times for Grenache and 7 times for Carignan) and then with 15% (5 times for Grenache and 7 times for Carignan) fresh model wine-like solutions until no further extraction of skin polyphenols could be observed (Fig. 1B).

Centrifugation pellets recovered from the diffusion solutions and "washed" skins were stored at -80 °C for further polyphenol analysis.

Statistical analysis

Statistical analysis was performed using Statistica software. The results obtained were assessed by factorial and one-way ANOVA analysis followed by a Tukey Test. Principal Component Analysis was performed on the AIS compounds (monosaccharides and amino acids) to assess the differences between varieties.

RESULTS AND DISCUSSION

3.1.Grape skin cell walls Alcohol Insoluble Solids (AIS)

AIS amounts in skins and their composition in monosaccharides and amino acids are reported for both variety and the four modalities (Fig. 1) in Table 1. No significant differences were observed between samples with respect to their content of AIS (mg AIS/g fresh skin) and the total content of neutral and acidic monosaccharides. For a given variety, these results are in accordance with those obtained previously on Syrah grapes 21 : total amounts of skin AISs remained constant during ripening and only minor changes in their monosaccharide composition were observed. However, factorial ANOVA followed by Tukey Test indicated (Supplementary Data S3) a significant difference between varieties (Grenache vs Carignan) regarding their galactose, mannose, and glucose composition. A centered reduced principal component analysis (PCA) was done on the obtained data set (Fig. 2A).

Variables and individuals were represented on the first 2 principal components (PC 1-2), describing 70% of the variability in the data. Mannose, galactose and glucose variables were negatively correlated on axis 1 to rhamnose and fucose. The axis 2 represented arabinose and xylose. The clear separation between the two varieties confirmed the results of the factorial ANOVA. The AISs of Grenache skins were richer in mannose, galactose and glucose compared to Carignan.

The primary cell wall of grape skins is formed by cellulose microfibrils tethered to a hemicellulosic matrix and of a more soluble domain consisting of pectic polysaccharides.

Hemicellulosic polysaccharides are mainly xyloglucans, which account for about 10 % of the wall polysaccharides. Pectic polysaccharides are homogalacturonans (HG, smooth regions of pectins), rhamnogalacturonans I (RG-I) carrying side chains of arabinose and arabinogalactans (hairy regions of pectins) and rhamnogalacturonans II (RG-II). They are embedded within the primary cell wall cellulose-xyloglucan framework and in the middle lamella 15 . As expected, galacturonic acid (from pectins) and glucose (from cellulose and hemicellulose) were the major sugars in skin AISs. Other neutral sugars were arabinose, galactose and rhamnose (from pectins), along with minor contents of xylose and fucose (from hemicellulosic polysaccharides) [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF] . The higher glucose content found in the Grenache may then indicate higher cellulose/hemicellulose content in this variety. The calculation of different specific ratios between neutral sugars has been proposed to characterize cell wall polysaccharides [START_REF] Ducasse | Effect of Macerating Enzyme Treatment on the Polyphenol and Polysaccharide Composition of Red Wines[END_REF] . Only the arabinose/galactose ratio differed between the two varieties (Table 1).

This ratio is characteristic of the PRAGs-like structures (polysaccharides rich in arabinose and galactose) [START_REF] Vidal | The polysaccharides of red wine: total fractionation and characterization[END_REF] . It was higher in Carignan than in Grenache AISs, indicating different compositions in the insoluble PRAGs of the hairy regions of pectins. The degree of esterification (DE) of skin cell wall pectins ranged from 55 to 85% (Table 1), which is consistent with that found for other grape varieties harvested at maturity [START_REF] Ortega-Regules | Differences in morphology and composition of skin and pulp cell walls from grapes (Vitis vinifera L.): Technological implications[END_REF] . The high deviations found for a given sample did not reveal any significant impact of variety or berry size and maturity. Amino acids of cell wall insoluble proteins represented from 8 to 12% of skin AISs (Table 1).

These amino acids belong to network of structural proteins. In general, our results indicated higher amino acid/monosaccharide ratios for Carignan than for Grenache. A factorial ANOVA followed by Tukey Test (Supplementary data S3) indicated an impact of both the variety and the maturity (deg + vs deg -modalities). The AIS of the Carignan skins were significantly richer in amino acids than those of the Grenache and deg + modalities were richer than deg -ones. Centered reduced PCA is shown on Fig. 2B. Variables and individuals were represented on the first 2 axes (1-2) that described 97% of the variability. All the variables were positively correlated on the axe 1 (90% of the variability).

Fresh skin polyphenol composition.

The polyphenol compositions in the fresh skins (Initial Skin Polyphenols, ISP) of the different Carignan and Grenache modalities were determined using UV-visible spectrophotometry (TPI, TRP), HPLC-DAD (total free anthocyanins) and HPSEC-DAD chromatography. UV-visible spectrophotometry and HPLC-DAD data showed differences between varieties, with significantly higher total polyphenol and anthocyanin contents in Carignan skins than in Grenache ones (Table 1).

They also evidenced a different composition, related to the respective proportions of non-acylated and p-coumaroylated anthocyanins, further discussed later. HPSEC analysis (Fig. 3) was used to determine the concentrations and the size distributions of polymeric tannins (aDP > 3). HPSEC profiles evidenced three main populations, eluted between 13 and 21 min. The first one corresponds to polymeric tannins with aDP > 3, the second one (maximum A280 at 19 min.) to oligomeric tannins (dimers and trimer) co-eluted with anthocyanins and the third one (maximum A280 at 20 min) to nonacylated anthocyanins. Polyphenols eluted after are lower molecular weight compounds. The concentrations and size distributions of polymeric tannin were similar in all cases (Table 1). Thus, the main differences between the two varieties and between the deg + and deg -modalities for a given variety were their anthocyanin content and anthocyanin/polymeric tannin ratios. The size of the berries (vol + , vol -) had no significant impact. Berry size may affect the skin to juice ratio and then their concentration in wines but also the concentration of secondary metabolites in skins [START_REF] Wong | Combined physiological, transcriptome, and cis-regulatory element analyses indicate that key aspects of ripening, metabolism, and transcriptional program in grapes (Vitis vinifera L.) are differentially modulated accordingly to fruit size[END_REF] . Such an impact on skin composition was not observed in our experimental conditions.

Polyphenol diffusion kinetic in wine-like maceration conditions.

Polyphenol diffusion in wine-like conditions was followed first by UV-visible spectrophotometry. The ethanol content in the solvent was progressively increased from 0 to 15% in 11 days to mimic solvent change during fermentation and maceration (Fig. 1B). However, unlike the conditions in red winemaking, samples were kept under constant stirring, i.e. under forced convective conditions in the liquid. Thus, the mass transfer is expected to be governed by the internal diffusion of polyphenols from within the skins to the solid-liquid boundary layer and to be faster than in winemaking [START_REF] Setford | Mass transfer of anthocyanins during extraction from pre-fermentative grape solids under simulated fermentation conditions: effect of convective conditions[END_REF] . In the present work, results have been reduced to the same mass of fresh skin (1 g) to compare the different modalities. TPI and TRP analysis indicated similar diffusion kinetic for both varieties and all modalities (Fig. 4). A large and rapid increase in total polyphenols and red pigments was observed during the first few hours of the diffusion, up to a maximum reached after 30 hours, which corresponds to 5% ethanol (Fig. 4B and4D). After that, a gradual decrease in TRP continued until the end of the experiment. This decrease was concomitant with a decrease in anthocyanins (measured by HPLC-DAD, Table 2). It may have several origins: i) degradation into low molecular weight compounds such as vanillic and syringic acids; ii) involvement in chemical reactions leading to the formation of both colorless or pigmented derived compounds with different molar extinction coefficients [START_REF] Cheynier | Structure and properties of wine pigments and tannins[END_REF][START_REF] Fulcrand | Phenolic reactions during winemaking and aging[END_REF] ; iii) involvement in physico-chemical interactions with other skin soluble components in the medium (polysaccharide, proteins) [START_REF] Bindon | Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components[END_REF][START_REF] Springer | Pathogenesis-Related Proteins Limit the Retention of Condensed Tannin Additions to Red Wines[END_REF] or solubility losses, leading to precipitation; iv) re-adsorption by solid parts. In parallel, total polyphenols also decreased for all Grenache modalities, but to a much lower extent, whereas a pseudo-plateau value was observed for Carignan ones. As anthocyanins present a higher extinction coefficient than flavanols at 280 nm, their decrease may hide the extraction of other polyphenols as quantified by UV spectrophotometry [START_REF] Boulet | Ultraviolet spectroscopy study of phenolic substances and other major compounds in red wines: relationship between astringency and the concentration of phenolic substances[END_REF] . Thus, after 30 h extraction, TPI profiles reflected equilibrium between polyphenol extraction from skins and anthocyanin decrease. HPSEC analyses were also performed at the end of three different steps of the diffusion experiment (0, 5 and 15% ethanol) to follow the extraction of polymeric tannins. These steps were chosen based on the analyses performed at the end of all steps (t0 to t5) with the vol + deg + modalities of the two varieties. Since the volume had no impact, results detailed in Fig. 5 are those obtained with vol + deg + and vol + deg -only. Polymeric tannin concentrations in the diffusion solutions increased for all Carignan modalities until the end of the 15% ethanol step (t5), but only very slightly after the 12% one. By contrast, after a maximum reached at 5% ethanol, a slight decrease of polymeric tannins was observed for all Grenache modalities, more pronounced for the deg -than the deg + ones.

Anthocyanin and tannin extraction: impact of variety, berry maturity and size

In wine-like maceration conditions, the concentrations of anthocyanins and tannins will be dependent on their extraction from skins but also from losses, which may be induced by chemical or physicochemical mechanisms. Maximum and final TRPs, anthocyanins and polymeric tannins concentrations in the diffusion solutions are reported for all modalities in Table 2 and compared to their concentration in the fresh skin 60% acetone extracts.

Anthocyanin extraction strongly differed between the two cultivars. At their maximum in the diffusion medium, the % TRP extracted were twice higher for the Grenache modalities (61-75%) than for the Carignan ones (28-32%). So, similar concentrations of red pigments were obtained by diffusion for the Carignan vol + deg -and the Grenache vol + deg + modalities whereas anthocyanin contents in the Carignan vol + deg -skins were twice higher. The % of extracted anthocyanins was calculated for non-acylated and p-coumaroylated anthocyanins (Table 2). An ANOVA analysis showed that they were significantly different between the two varieties, but not between different berry size and maturity for a given variety. Besides, soluble anthocyanins were mainly the nonacylated ones, from the beginning to the end of the experiment. The coumaroylated/non acylated anthocyanin ratios varied between 0.3 (deg + ) and 0.5 (deg -) in the Carignan skins, and between 0.15 (deg + ) and 0.25 (deg -) in the Grenache ones. In solution, these ratios fell to values between 0.05 and 0.1. These results are consistent with previous ones [START_REF] Fournand | Accumulation and extractability of grape skin tannins and anthocyanins at different advanced physiological stages[END_REF][START_REF] Favre | Selectivity of pigments extraction from grapes and their partial retention in the pomace during red-winemaking[END_REF] that showed a poor extraction of pcoumaroylated anthocyanins in both simulated extraction experiments and winemaking for other grape varieties. However, these structural features alone did not account for the differences observed here between Carignan and Grenache, as lower extraction were also found for non-acylated anthocyanins in Carignan. According to Ortega-Regules et al. [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF][START_REF] Ortega-Regules | Differences in morphology and composition of skin and pulp cell walls from grapes (Vitis vinifera L.): Technological implications[END_REF] , higher anthocyanin extractability could be associated with low concentrations in galactose, glucose and mannose in the skin AISs, along with a low DE of pectins. These results, obtained from the comparison of four varieties (Cabernet-Sauvignon, Syrah, Merlot and Monastrell), are not in total agreement with those obtained here: Carignan had in fact lower galactose content than Grenache (and higher arabinose/galactose ratios), but also lower glucose and mannose contents (cellulose, hemicelluloses). This suggest that differences in the insoluble PRAGs of pectins could be of importance in anthocyanin extractability.

Polymeric tannins extraction at maximum only represented a small proportion of skin polymeric tannins, as expected [START_REF] Ortega-Regules | A first approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability[END_REF][START_REF] Bindon | Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass[END_REF][START_REF] Fournand | Accumulation and extractability of grape skin tannins and anthocyanins at different advanced physiological stages[END_REF] . It was higher for Grenache (from 13 to 28%, depending on the modalities), than for Carignan (10 to 20%), especially for the deg + modalities (Table 2). It has been previously observed with Syrah that higher anthocyanin/tannin ratios in skins led to a higher extraction of skin tannins [START_REF] Kilmister | A role for anthocyanin in determining wine tannin concentration in Shiraz[END_REF] . This ratio was higher and statistically different (Tukey test) for Carignan, and among the Grenache modalities, differences were significant between deg + and deg -modalities (Table 1). It could be concluded from present results and as for Syrah that higher anthocyanin/tannin ratios favour the extraction of tannins for Grenache (deg + vs deg -modalities, Table 2). However, this was not the case for Carignan and was not verified when comparing the two varieties. Differences in the composition of AISs may have offset a positive impact of anthocyanins. Differences in tannin extractability are largely attributed to differences in the polysaccharide composition of skin AISs [START_REF] Hazak | The phenolic components of grape berries in relation to wine composition[END_REF][START_REF] Bautista-Ortín | The Extraction Kinetics of Anthocyanins and Proanthocyanidins From Grape To Wine[END_REF] and/or to their protein content, higher in Carignan than in Grenache. In addition to higher maximums in solution, tannin diffusion was faster for Grenache than for Carignan, and favored by increasing ethanol contents for the latter. This may be due to stronger interactions of tannins with cell walls, hydrophobic interactions and H-bonds being weakened by the ethanol concentration. In accordance with literature, the comparison of polymeric tannin size distribution in skins and in solutions (Fig. 3 and 5, supplementary data S2) indicated that the highest molecular weight tannins are not extracted in wine-like solvents [START_REF] Bindon | Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins[END_REF][START_REF] Fournand | Accumulation and extractability of grape skin tannins and anthocyanins at different advanced physiological stages[END_REF] . Indeed, skin polymeric tannins were eluted between 13 and 18.2 min., with a peak maximum around 15-16 min whereas extracted tannins were eluted between 15 and 18.2 min, with a maximum around 17-17.3 min.

After a maximum, a decrease in polymeric tannins measured by SEC (peak 1, Fig. 5) was observed for all Grenache modalities that reduced differences between varieties at the end of the maceration (Table 2). A much more pronounced decrease was also observed in all cases for red pigments. At the end of the diffusion, skins were recovered and the whole diffusion media clarified by centrifugation. Red-coloured pellets were present in all cases. Their visual observation showed the existence of two types of materials: one, located at the bottom of the centrifuge tube and having a fibrous-like appearance, and another, above, having a gel-like appearance. The exact nature of these deposits was not further investigated here and will be the subject of future work. Their presence indicated that either precipitation of soluble material from skin cells and degradation of some insoluble material leading to the presence of cell fragments/fibers occurred during our maceration experiments. This material was found in higher amounts in the Carignan modalities than in the Grenache ones (Table 2).

Polyphenols in these pellets (PSP, Fig. 1), were extracted and quantified. Results confirmed the presence of anthocyanins and tannins, related to precipitation or adsorption phenomena. They represented a small but non negligible part of skin polyphenols, and were in higher amount for Carignan. Red pigments in PSPs accounted for 7-9% of the initial skin ones for Carignan, to be compared to 3-5% for Grenache, with no clear incidence of berry maturity or size. Polymeric tannins accounted for 6-9% of the initial skin tannins for Carignan and for 3-6% for Grenache. HPLC-DAD analyses evidenced a preferential involvement of coumaroylated anthocyanins in the precipitates (Table 2), with coumaroylated/non-acylated anthocyanin ratios between 3.8 (deg + ) and 6.3 (deg -) in

Carignan PSPs and around 1 in Grenache ones. This can be compared to the same ratio in ISPs: within the range [0.30-0.50] for Carignan and [0.14-0.27] for Grenache. This indicated a lower solubility of coumaroylated anthocyanins and/or favored interactions with other constituents extracted from skins.

Whatever the variety or the modality, anthocyanins in PSPs did not explain the anthocyanin losses observed in solution during maceration (SSP at max versus SSP at the end). Considering first Carignan, losses in p-coumaroylated anthocyanins during maceration represented between 0.1 and 0.7 mg.L -1 and were then much lower than their content in PSPs (7.3 to 10.1 mg.L -1 ), whereas losses in non-acylated anthocyanins represented between 10 and 12.4 mg.L -1 and where much higher than their contents in PSPs (1.1 to 2.3 mg.L -1 ). For Grenache, p-coumaroyled anthocyanins in PSPs could be related to their precipitation from solution in all cases but the vol -deg + modality. In all cases and as with Carignan, losses in non-acylated anthocyanins (between 2.4 and 10.3 mg.L -1 , depending on the maturity) were much higher than their content in PSPs (0.07 to 0.2 mg.L -1 ). This indicated that there were also losses of anthocyanins related to chemical reactions. TRP at the end of the maceration experiment represented between 40 (deg + ) and 50% (deg -) of the maximum TRP value for the Grenache modalities (to be compared to 42 and 54 % for anthocyanins) respectively, and between 30 (deg + ) and 43% (deg -) for the Carignan ones (42 and 53 % for anthocyanins). Thus, anthocyanin degradation and/or the formation of non-pigmented derived compounds, such as those from anthocyanin-tannin adducts, were likely the predominant phenomenon in the observed decreases. In the Carignan, part of anthocyanin losses could be attributed to the formation of derived red pigments such as tannin-anthocyanin adducts.

As for coumaroylated anthocyanins, it is unclear whether tannins in PSPs were precipitated or adsorbed very quickly after their solubilisation or whether their presence was related to their interaction inside the cells with debris/fibers released during maceration. A slight decrease in tannin concentrations was observed during the experiment with the Grenache, not with the Carignan (Fig. 5, Table 2). As extraction was more gradual for this variety and analyses only carried out at the end of the various ethanol additions, extraction may have counterbalanced losses related to physicochemical mechanisms.

Residual polyphenols in skins after maceration

Skins after diffusion experiments were successively washed with new 0 and 15% ethanol wine-like solvents until total polyphenol extraction (Fig. 1B). These washings led to the recovery of so-called residual extractible polyphenols in wine-like solvents (RESP, Table 2). The latter represented from 7 to 12% of the initial skin anthocyanins for both variety and from 6 to 9% of the polymeric tannins. As solid/liquid diffusion is driven by a partition coefficient between the solid and the liquid phases, their extraction in new solvents indicated that an equilibrium between phases had been reached in our conditions at the end of the maceration. Finally, residual polyphenols in skins (non-extractable skin polyphenols NESPs) were extracted in 60% acetone, as for ISPs and PSPs.

Only very small amounts of red pigments were recovered (Table 2). NESPs were mainly polymeric tannins (Table 2, Fig. 6), along with small amounts of oligomers. Polymeric tannins recoveries in NSEP were higher for the Carignan (between 24 and 40%) than for the Grenache (between 12 and 30%) and, within a given variety, about twice higher for the deg + (39 and 27%) than for the deg -( 24and 13%) modalities.

Similar HPSEC profiles were found for SSP and RESP (Fig. 6), whatever the variety and the modality. HPSEC also evidenced a wider size distribution of polymeric tannins in PSPs by comparison to diffusion media, related to the presence of higher molecular polymers, and strong differences between Carignan and Grenache, related to p-coumaroylated anthocyanins (supplementary data S4). As expected, NESPs were preferentially the highest molecular weight tannins in skins. A mass balance was performed with the whole results obtained from our diffusion/extraction experiments (Table 2, Fig. 6). Taking the sum of TRP in SSP (end of the maceration), PSP, ESP and NESP resulted in the recovery of only 45 to 64% of the initial skin red pigments for Grenache (to be compared to 19-36% of the anthocyanins) and of 31 to 51% for Carignan (25-28% of the anthocyanins), with no clear impact of the maturity (deg + vs deg - modalities). Although no fermentation was carried out under our experimental conditions, our results are consistent with those of Morel-Salmi et al. [START_REF] Morel-Salmi | Effect of flash release treatment on phenolic extraction and wine composition[END_REF] , who observed a drastic loss (around 70 %) of anthocyanins during fermentation when they compared anthocyanins present in the initial skins, the wines and the pomaces. Contrary to that observed with anthocyanins, higher tannin recoveries were found for the Carignan (72-74% for the deg + modalities and 53-56% for the deg -) than for the Grenache (60-70% for deg + and 33-38% for deg -), but as for anthocyanins, they were not complete.

Besides, HPSEC indicated an excess of A280nm in the range of oligomers and smaller polymers (17 -19 minutes) for all modalities except Grenache deg -. These elution times correspond to trimersheptamers according to Figure S2. This excess, along with the differences observed between TRPs and anthocyanins, agrees with the occurrence of chemical changes during maceration. The latter may affect anthocyanins as well as tannins alone. Indeed, even in the absence of fermentation and under conditions of protection against oxidation, cleavage and rearrangements reactions can also occur within tannins that does not imply anthocyanins [START_REF] Cheynier | Structure and properties of wine pigments and tannins[END_REF][START_REF] Morel-Salmi | Effect of flash release treatment on phenolic extraction and wine composition[END_REF][START_REF] Vidal | Changes in Proanthocyanidin Chain Length in Winelike Model Solutions[END_REF] . These chemical reactions may also occur within skins when the integrity of the berries is broken. The quantification of polymeric tannins by HPSEC was based here on A280nm measurements and was done in equivalent epicatechin. The molar absorptivity of tannins differs as a function of their degree of polymerization, even if their chemical nature is not changed [START_REF] Kennedy | Analysis of proanthocyanidin cleavage products following acidcatalysis in the presence of excess phloroglucinol[END_REF] and can also be modified by chemical reactions. As SEC separation is based on the size, polymers with the same size but different chemical features (including different molar extinction coefficients) are coeluted, which may induce a misquantification.

Besides chemical changes, it must be considered that the same extraction procedure was performed on fresh skins, precipitates and skins after maceration and washings, whereas these samples have different characteristics. Changes in cell-wall structures during the maceration process (11 days), leading to different permeability and solvent accessibility, as well as to stronger physicochemical interactions than those observed in fresh skins or possibly the formation of covalent bonds, may have resulted in a lower extraction at the end (NESP vs ISP). The fact that both anthocyanins and tannins total recovery differed between the Carignan and Grenache varieties and for tannins, between the deg + and the deg -modalities, is of interest and likely indicated structural differences in cells/cell walls in the initial skins and/or different changes during maceration that deserve to be further explored.

Conclusion

The diffusion results obtained under model conditions, as well as the analyses carried out on the precipitates observed at the end of maceration and on the residual skins, made it possible to highlight differences between the two varieties studied and between berries with different levels of maturity.

The impact of berry size was much less evident. In agreement with the literature, these differences concerned first the extraction of anthocyanins and tannins. The former was proportionally much more important in Grenache than in Carignan. This could be partly explained by the higher proportion of p-coumaroylated anthocyanins in Carignan skins but the extraction of non-acylated anthocyanins also was lower for this variety. The extraction of tannins was also slightly higher but above all much faster in Grenache than in Carignan, and higher for the deg + modalities. No decisive impact of the anthocyanin-to tannin ratio on the tannin final contents in solution was observed.

Carbohydrate and amino-acid analysis of skin AISs evidenced also differences in composition between the two varieties, but not between the different modalities (especially ripeness). These differences were related to glucose (cellulose/hemicelluloses), in higher content in Grenache than in Carignan, and to different arabinose/galactose ratios reflecting different structures of PRAGs. Higher protein contents were found in Carignan skin AISs. However, compositional analyses on AISs do not provide any structural information on the initial polymeric network in skin cell walls. This information, combined with a better knowledge of its changes during maceration and a better understanding of the part played by the chemical reactivity of tannins and anthocyanins in solution or in skins, is needed to understand the impact of variety and/or berry maturity on tannin and anthocyanin extractability.

Table 1. Alcohol Insoluble Solids contents (AIS) and their monosaccharide and amino acid compositions, and polyphenol contents of the four modalities of Carignan and Grenache initial skin berries. Results are expressed in mg/g fresh skin or AIS. Different letters indicate significant differences between samples for a given parameter (Tukey's test for p < 0.05). DE (%) is the percentage of methylesterification.

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. PCA analysis of the variables and individual distribution regarding monosaccharides constitutive of polysaccharides (A) and amino acids constitutive of proteins (B) in the skin AISs of Carignan and Grenache different modalities.

Figure 3 .

 3 Figure 3. Initial polyphenols contents in the skins of the different modalities (vol + deg + , vol - deg + , vol + deg -and vol -deg -) by HPSEC. Carignan: car; Grenache: gre. Peak 1: polymeric tannins with aDP > 3; peak 2: oligomeric tannins (dimers and trimer) co-eluted with acylated anthocyanins; peak 3: non-acylated anthocyanins.

Figure 4 .

 4 Figure 4. Polyphenols diffusion during skin maceration experiments in wine-like solvents, followed by UV-visible spectrophotometry. TPI: Total Polyphenol Index (A280 nm). TRP: Total Red Pigments (A520 nm). Arrows indicate the different ethanol additions. A) Carignan, TPI; B) Carignan TRP; C) Grenache TPI, D) Grenache TRP.

Figure 5 .

 5 Figure 5. HPSEC analysis of polyphenols at the end of different steps of the diffusion experiments. Peak 1: polymeric tannins; Peak 2: di-trimers and anthocyanins, Peak 3: anthocyanins. Integration of the 3 main peaks of the HPSEC spectra at the end of the first (t0, 0% ethanol), second (t1, 5% ethanol) and last (t5, 15% ethanol) steps of the maceration experiment for the vol + deg + and vol + deg -modalities of the Carignan (A) and the Grenache (B) varieties. Comparison of the HPSEC profiles of the vol + deg + (C) and vol + deg -(D) modalities of Grenache and Carignan at the end of t1 and t5.

Figure 6 .

 6 Figure 6. Comparison of the HPSEC profiles of the different phenolic extracts for the different Grenache and Carignan vol + deg + and vol + deg -modalities. A) Carignan vol + deg + B) Carignan vol + deg -C) Grenache vol + deg + D) Grenache vol + deg -. ISP: initial skin polyphenols; SSP: soluble skin polyphenols at the end of the maceration experiment; PSP: Precipitated skin polyphenols at the end of the maceration experiment; RESP: residual extractable skin polyphenols in wine-like solvents (extraction in 60 % acetone solvent); NESP: non-extracted skin polyphenols in wine-like solvents; SUM = SSP + PSP + RESP + NESP.

  Figure 1

  Figure 2

Table 2 .

 2 Polyphenol extraction. Values represent concentrations reduced considering 1 g fresh skin in 42 mL solvent (in mg/L eq. malvinin-3-O-glucoside for anthocyanins and mg/L eq. epicatechin for tannins). ISP: initial skin polyphenols (60% acetone); SSP: soluble extracted skin polyphenols (at maximum and at the end of the maceration experiment); PSP: Precipitated skin polyphenols; RESP: residual extractable polyphenols in wine-like solvents; NESP: non extracted polyphenols (60% acetone). Non-acyl. (non-acylated) and p-coum. (p-coumaroylated) anthocyanins. Different letters indicate significant differences between samples for a given parameter (Tukey's test for p<0.05).

	Carignan

Acknowledgments

This work was carried out as part of "Interfaces" flagship project, publicly funded through ANR (the French National Research Agency) under the "Investissements d'avenir" program with the reference ANR-10-LABX-001-01 Labex Agro and coordinated by Agropolis Fondation under the reference ID 1603-001. The authors would also like to thank their colleagues from the Pech Rouge Experimental Unit for their help in harvesting and sorting the berries and their colleagues from the UMR SPO for their participation in the preparation of the samples.