Corentin Durand

Maxime Berthe

Younes Makoudi

Jean-Philippe Nys

Philippe Caroff

Bruno Grandidier
email: bruno.grandidier@isen.iemn.univ-lille1.fr

Renaud Leturcq

Persistent enhancement of the carrier density in electron irradiated InAs nanowires

Introduction

Semiconductor devices are more and more relying on the integration of nanostructures to enhance their optical and electronic performances. In order to comply with a high level of quality and reliability, the controlled positioning of the nanostructures in the devices and the subsequent inspection of their integrity during the manufacturing processes require the use of high resolution analytical and imaging techniques. Devices consisting of single semiconductor nanowires (NWs) are generally not an exception to this rule. For example, the inspection, localization and connection of nanowires with electrical electrodes are hard to achieve without an electron beam [1]. However, the excitation of materials with incident electrons can stimulate a wealth of reactions that are known to modify the material properties [2,[START_REF] Vajda | Anisotropy of electron radiation damage in metal crystals[END_REF]. For years, the tolerance of materials and devices to a beam of electrons has indeed been of great concern in many technological applications, because electrons are known to produce defects and generate disorder in materials [4,5,6].

While outstanding performances or physical effects have been recently demonstrated with semiconductor NWs [7,8,9,10,11], a detailed study of the electrical modifications that are caused by electron beam irradiation is still missing. For example, contact processing or contact through a retrofitted probe are usually achieved on InAs NWs with the help of a scanning electron microscope (SEM) working with an energy beam in the range of 5 to 20 keV [12,13]. Within this energy range, the electrons interact with the entire section of the NWs [14]. As it is well established that electron irradiation of InAs bulk material causes a Fermi level pinning high in the conduction band [15], questions arise to determine to what extend an electron beam might change the electrical properties of InAs NWs.

A serious impediment to study the direct impact of electron irradiation on the NW conductivity resides in the presence of remote charges in the NW environment, that can electrostatically control their conductivity [16]. In order to circumvent this drawback, sophisticated characterisation techniques must be developped, based, for example, on multiple probe scanning tunneling microscopy (STM), that enable arbitrary arrangements of probe electrodes on one-dimensional nanostructures [17,18]. Taking advantage of this technique, we therefore investigate electrical transport in non intentionnaly doped InAs NWs that were subject to electron irradiation. Upon irradiation, we find a significant and reproducible decrease of the resistivity, leading to NWs with a metallic-like character. This phenomenon is persistent in ultra high vacuum (UHV) and the NWs barely recover their initial resistivity once the NWs have been exposed to air. While the transport measurements show a degradation of the mobility with the irradiation, this effect is largely counterbalanced by a strong increase of the electron concentration. Study of the NW resistivity dependence on the surface chemistry provides a compelling evidence for a conduction dominated by accumulated electrons. Such a localized increase of the electron density in the NWs, that is supported by numerical analysis, allows discriminating between the contribution of surface and bulk charged defects.

Methods

[0001]-oriented wurtzite InAs NWs were grown from gold particle seeds using gas source molecular beam epitaxy. Starting with a short InP stem grown at 410°C on InP(111)B substrates, InAs NWs were subsequently obtained by direct switch from P 2 to As 2 molecular fluxes. The NWs are of very high crystallographic quality, with only a very limited number of single stacking faults along their growth axis, as shown previously [19]. Two types of geometry were electrically investigated: interconnected NWs with conventional planar technology and free-standing NWs. For the first ones, NWs were deposited onto a degenerately-doped Si wafer with a 225 nm of thermally-grown SiO 2 layer and selected for contact processing with a scanning electron microscope at low magnification and with a short integration time. After spin-coating the sample with an electron-beam sensitive polymer, four contacts are defined along the nanowire using electron beam exposure. The oxide on the contact area was etched in a (NH 4 ) 2 S x solution just before evaporation of Ti/Au electrodes (10/150 nm thick) [20]. This step was followed by a thorough lift-off to get NWs with clean oxidized sidewalls.

As to the second type of geometry, the as-grown NWs were kept on the InP substrate. The NW mean diameter was 70 ± 9 nm, but NWs with smaller and bigger diameters could sporadically be produced. All the experiments were carried out with the same setup, consisting of a SEM and a multiprobe STM (Nanoprobe, Omicron Nanotechnology), that was operated with a control system able to run the four STM tips independently (Nanonis multiprobe control system, SPECS). The electrical measurements were performed in UHV (base pressure lower than 5x10 -10 mbar) and in the dark, always with the electron beam turned off.

In order to investigate how the surface chemistry modifies the Fermi level pinning, three different surface treatments were applied. The NWs were either left in air for several days to ensure a complete oxidation of their sidewalls. Or, they were passivated with an ammonium sulfide solution to produce a chemisorbed S layer on their surface and, then, quickly loaded into the Nanoprobe system. Indeed this chemical strategy has been shown to efficiently remove the native oxide on the InAs (001) surfaces, offering greater chemical stability to electronic devices [21]. Finally, NWs were also capped with a thin layer of As at the end of the growth and subsequently transferred to the Nanoprobe system. The evaporation of the capping layer was achieved at 350°C, monitoring the As desorption with mass spectrometry. This preparation was always achieved prior to the SEM inspection of the NWs and their subsequent electrical characterization. Apart from the existence of small Au clusters, that are caused by the gold diffusion along the NW sidewalls from the seed particle during the evaporation of the As capping layer, such a method allows to restore the initial crystalline surface structure of the NWs [22].

Results and discussion

The resistance of the NWs and its change with electron irradiation were first investigated for interconnected InAs NWs, with native-oxide covered sidewalls. A CCD camera was used to position the STM tips on metallic pads, thus avoiding the exposure of the NWs to the e-beam, as shown in Figure 1a andb. Two-terminal current-voltage characteristics are found to be linear, and clearly reveal a decrease of the resistance once an InAs NW have been exposed to a 5 keV electron beam for 0.35 s (Figure 1c), in agreement with the litterature [23]. Periodic measurements of the resistivity over 48 hours (events 1 to 5) show that the NW retains its low resistivity after irradiation (Figure 1d). A subsequent exposure to air for four hours allows us to partially restore a high resistivity (event 6). But, as soon as the NW has been again exposed to the electron beam, the resistivity returns to its low value (event 7).

As we can not rule out that the change of the resistivity could be caused by charges trapped in the surroundings of the NWs, since dielectric materials are known to efficiently retain charges in UHV [24], similar free-standing InAs NWs with native-oxide covered sidewalls were characterized by the four-probe method. In such a configuration, four STM tips were brought into electrical contacts with the NW using the distance regulation of the STM control system under the monitoring of the SEM (Figure 2a). Tips 1 and 3 were connected to the seed alloy particle and the base of the NW, respectively. They were used as source and ground electrodes for driving the current through the NW, while the substrate was kept floating. Tips 2 and 4 were the potential probes. In most cases, the I(V) curves between a single tip and the substrate, held at ground, show an ohmic behaviour (see lower inset of Figure 2b). When a current is passed through a NW, the potential drop measured between the inner probes is found to be linearly proportional to the current intensity (Figure 2b). The gradient of the curves yields the four-probe resistance of the NWs for a given separation between the inner probes. By moving the inner tips towards the center of the NW, a set of V(I) characteristics was acquired for different tip separations L. From these V(I) curves, we obtain the resistance R NW that is found to linearly increase with L, as shown in the upper inset of Figure 2b. The slope yields a resistance per unit length R NW /L of 6.3 kΩ/µm. We performed similar measurements on an ensemble of free-standing NWs, that were all subject to irradiation, since their connection with the STM tips can not be managed without the use of the SEM. We also measured the resistance per unit length of interconnected NWs, that were not irradiated. The resistances per unit length of all the NWs were then compared.

Figure 3a clearly reveals a decrease of the resistance, when the NWs have undergone an electron irradiation. Such a behaviour is consistent with the one observed in the two-terminal experiments. It unequivocally demonstrates that the increase of conductivity is caused by the action of the incident electrons impinging on the NW. The high conductivity of the irradiated NWs is further evidenced by four-probe measurements performed at low temperatures (Figure 3b). For these experiments, the data points shown in Figure 3 at different temperatures are usually measured on different nanowires, due to the difficulty to keep the tips connected to the same nanowire while changing the temperature. Remarkably, the variation of the resistance per unit length shows no particular sign of dependence on the temperature, indicating a metallic nature for the irradiated NWs. This is in clear contrast with the typical semiconductor behavior found for a non-irradiated NW (inset of Figure 3b), where a threefold variation is measured for R NW /L in the temperature range 300K-120K.

In order to quantify the carrier concentration in the conduction band, the field effect mobilities were estimated from gate sweeps of the source-drain current I ds at a fixed source-drain voltage V ds of 50 mV (Figure 4). The unmodified InAs NWs exhibit a typical n-type response with increased current at more positive back-gate voltage V g (spectrum s1). Conversely, upon irradiation, the NWs can not longer be pinched off at V g higher than -20V (spectrum s2), consistent with the strong metallicity of the irradiated NWs deduced from the four-probe measurements. Then, after air exposure (spectrum s3), the resistance increases again, but the transconductance (g = dI ds /dV g ), that is related to the field effect mobility [25], gets worse.

As the change of the transconductance reveals a decrease of the mobility after irradiation, the occurence of a low resistance in the interconnected NWs as well as in the free-standing NWs is the signature of an increase of the electron concentration to compensate for the charges trapped at the NW surface or in the NW. Assuming a diffusive transport [26] and taking the value of the mobility measured after air exposure as an upper bound, we estimated the carrier concentration from the inverse of the product between the elementary charge, the measured mobility and resistivity. For the NW with a diameter of 40 nm, we find an electron concentration of 1.2x10 19 cm -3 (Figure 4a). As to the second NW shown in Figure 4b, which has a smaller diameter, the estimated carrier concentration is even larger, reaching 1.4x10 19 cm -3 . Such carrier concentrations demonstrate a stabilization of the Fermi level quite high in the conduction band.

Although this carrier concentration is in agreement with the huge electron concentration found in proton-irradiated bulk InAs (1.8x10 19 cm -3 ) [15], InAs surfaces have been shown to exhibit a surface electron accumulation layer that can be quite deep depending on the surface chemistry [27]. In order to determine whether the Fermi level pinning arises from the formation of surface or bulk charged defects, the dependence of the NW resistivity on the surface chemistry was also investigated. In case of a stabilization of the Fermi level due to bulk charged defects, we would expect negligible modifications of the NW resistivity with the surface chemistry. As evidenced in Figure 3a, a change of the surface chemistry for irradiated NWs with similar diameters causes a significant variation of their resistance per unit length. In order to get insight into the electron distribution along the radial axis of the NWs, onedimensional numerial simulations have been performed [33]. To model the numerous interface states on the NW sidewalls, we therefore assumed a positive charge density n i at the NW surface and varied it in the range 10 11 to 10 15 cm -2 (surface atomic density of InAs{10-10}) to account for the generation of charged defects by the electron beam. Figure 5 shows the calculated variation of the spatially average electron concentration n av with n i for InAs NWs with a diameter of 26 and 40 nm [34]. For values of n i in the range of 10 12 cm -2 , that are typical of surface/interface state densities obtained for non-irradiated InAs [23,35], we find n av consistent with the measured electron concentrations, that are marked in Fig. 5 by the horizontal dashed segments at positions s1. After irradiation, n i increases and causes an increase of n av to fulfill the condition of charge neutrality in the NW (dashed segments s2).

Interestingly, the resulting electron concentration is still larger for the smaller diameter, in agreement with charges accumulated at the surface, while bulk charge accumulation would lead to the opposite trend since quantum confinement implies a lower density of states for smaller diameter. In UHV, our estimation of the electron concentration indicates an interface charge density slightly higher than 10 13 cm -2 . Then, the release of the NWs to the air reduces n i (dashed segments s3). Such effect is likely related to the partial annihilation of charges trapped on the NW sidewalls in air, while a structural disorder induced by irradiation still exists and causes the observed decrease of electron mobility.

More remarkably, an increase of the electron concentration after irradiation takes along with a narrowing of the accumulation layer, as shown in the inset of Figure 5. In this case, transport mainly occurs close to the NW surface, where the surface charged defects seriously scatters the charge carriers and reduce their mobility. Such a result is consistent with a surfaceinduced change of the resistance observed for InAs NWs with different surface chemistries. It indicates that a change of the surface state density by impinging energetic electrons is sufficient to explain the huge increase of the electron concentration in the NWs. Therefore the Fermi level in the NWs is not stabilized by charged defects trapped in the bulk of the NWs, but it stays pinned at the NW surface after irradiation. Finally, we would like to point out that the observed decrease of the resistivity under electron irradiation holds for InAs, because the stabilization of the Fermi level in this material occurs far above the bottom of the conduction band [36]. This level is reached when the concentration of defects becomes very high, causing the Fermi level to be pinned by the fulfillment of the condition for charge neutrality. For most of the other semiconductor materials, the charge neutrality level resides in the band gap [36].

The generation of defects thus leaves these materials with a semiconductor character, in agreement with the electrical behaviour recently found for a few irradiated semiconductor nanostructures [37,38].

Conclusions

In conclusion, we have shown that electron irradiation can significantly change the electrical properties of InAs NWs. At 5 keV, the material modifications are predominantly restricted to the surface of the NWs, involving a deepening of the electron surface accumulation to compensate the surface charged defects. We infer that surface charged defects induced by electron irradiation should occur in all types of semiconductor nanomaterials, causing more or less noticeable changes in their transport properties as a function of the defect concentrations, energy levels and charge states. Such a generation of defects in semiconductor nanostructures could be helpful to guide our understanding of their electronic properties, when they are interfaced with other materials, for example in the creation of heterostructures and metalsemiconductor contacts. As to InAs material, the defect-induced electrostatic effect is significant and pleads for the need of a protective layer. For example, InAs NWs with oxidized or sulfur-passivated sidewalls could be protected with a thin shell of amorphous As material, as the one used to cap the bare InAs NWs with clean and crystalline sidewalls. After the SEM inspection and probe positioning, the protective As capping layer could be desorbed by direct current heating, leaving the surface free of radiation-induced defects.

  The highest resistance is obtained for the NWs with clean and crystalline sidewalls, whereas the S-passivated NWs are the most conductive NWs. Remarkably, such a trend is consistent with the depth of the accumulation layers measured for non-irradiated InAs surfaces prepared with different chemical treatments [28]. Interestingly, it has been shown, theoretically and experimentally, that In adatoms on different InAs surfaces act as donors and thereby cause the formation of a charge accumulation at the surface [29,30]. As electron irradiation of the GaAs(110) surface produces vacancies consisting of Ga-As pairs [31] and, Langmuir evaporation of anions on similar surfaces is known to occur at room temperature [32], we suspect the irradiation of the InAs sidewalls to substancially increase the initial concentration of isolated In adatoms on the clean and cristalline sidewalls of the bare InAs NWs. Assuming the binding of an In adatom to two As atoms and one In atom, the electron counting rule yields an occupied In dangling bond. As the related state is resonant with the conduction band [29], the formation of In adatoms causes a partial filling of the conduction band, resulting in the creation of positively charged defects on the sidewalls of the NWs. Despite the scarcity of data for the oxidized and S-passivated irradiated InAs surfaces, the desorption of anions and the formation of cation adatoms are also very likely on these surfaces under irradiation and could lead to a similar surface accumulation layer, depending on the atomic configuration of the In adatoms.

Figure 1 .

 1 Figure 1. (a) Large scale SEM image of the sample geometry used to characterize the electrical transport in interconnected InAs nanowires. Two STM tips are plugged to the source and drain electrodes (scale bar: 300 µm). (b) SEM enlarged view of the InAs NW in electrical contact with four electrodes (scale bar: 1µm). (c) Two-terminal current-voltage characteristics of an InAs NW with a diameter of 40 nm, measured before and after a 5 keV irradiation for a dose of 9.2x10 17 e.cm -2 . (d) NW resistance measured as a function of the treatments, that are described in the main text, for a period of time of 72 hours.

Figure 2 .

 2 Figure 2. (a) SEM image of free-standing InAs NWs grown on an InP(111)B substrate. The NW in the centre is contacted with four W tips, that are used either as sourcing electrodes or potential probes. Electron beam energy: 5 keV. Scale bar: 150 nm. (b) V-I curves measured with the four probe method for different spacings L between tips 2 and 4. The length L is given to the right of the graph. Upper inset: Resistance measured from the V-I measurements as a function of L. Lower inset: I-V curve measured between tip 2 and the substrate.

Figure 3 .

 3 Figure 3. (a) Resistance per unit length R NW /L measured at room temperature as a function of the InAs NW diameter for interconnected InAs NWs (filled symbols) and free-standing and irradiated InAs NWs (open symbols). (b) Temperature dependence of R NW /L for irradiated NWs. Inset: Temperature dependence of R NW /L for a non-irradiated NW with a diameter of 100 nm.

Figure 4 .

 4 Figure 4. Source-drain current versus gate bias measured in ultra high vacuum for two InAs NWs, with a diameter D, as a function of the treatment: s1) before irradiation, s2) after an irradiation at 5 keV and doses of a) 9.2x10 17 e.cm -2 and b) 4.6x10 17 e.cm -2 , s3) after an air exposure of 4 hours.

Figure 5 .

 5 Figure 5. Simulated average electron concentration n av as a function of the density of surface states n i for two InAs slabs with thickness D of 26 and 40 nm respectively. The measured

Acknowledgments

This study was financially supported by the EQUIPEX program Excelsior, and C. D. Y.M., R.L., P.C. respectively acknowledge the financial support of the DGA, CEA-LETI and ANR through the project number ANR-11-JS04-002-01. We thank T. Melin, K. Smaali and X.

Wallart for discussions.


F Bonnet

R Schweins

F Boué

E Buhler

Suppression of Aggregation in Natural-Semiflexible/Flexible Polyanion Mixtures, and Direct Check of the OSF Model using SANS

Keywords: numbers: 82.35.Rs, 61.12.Ex, 87.15.-v

Suppression of Aggregation

("polyelectrolyte peak") signalling a privileged repulsion distance between chains; this peak broadens and moves to higher scattering vectors q as the concentration is increased [5]. On the contrary, a major debatable point concerns the aggregation behaviour observed at small q and correlated to the slow mode measured by dynamic light scattering [6,7]. Indeed, it is surprising that two macromolecules with like charge attract while they repel at shorter distances. This occurs reproductively on different charged biopolymers, including DNA, proteins and polysaccharides when ionic strength, I, is low [6,7]. During the last decade, there has been exciting debates in the "polyelectrolyte community" to elucidate this phenomenon.

In particular, a well-accepted model developed by Ray and Manning and based on an attractive force between polyions mediated by the sharing of condensed counterions is generally used to interpret these experimental facts [8]. Beyond its peculiarity, this low-q aggregation signal has the disadvantage of masking a length domain necessary for any determination of the chain persistence length, to which we come now. Indeed, another question concerns the rigidity induced by inter like-charged monomer repulsion; this is quantified by an "electrostatic persistence length", L e that depends on I, which is due to counterions as well as external salt. There has been some theoretical debate about whether L e varies like  -1 or  -2 , where  -1 (~I -1/2 ) represents the Debye length [1].

These theories describe the polyelectrolyte as a "wormlike" chain with a total persistence length, L T = L 0 + L e , where L 0 is the intrinsic persistence length of the uncharged polymer. For semi-rigid polyelectrolytes, with L 0 easily larger than L e , Odijk [9], and Skolnick and Fixman [10] found:

2 2 4 b l L B e   for l B /b<1 (OSF relation, valid if L T >>1) (1)
The Debye length is  -1 = (4l B c f ) -1/2 (the Bjerrum length l B =7.13 Å in water), c f = c+2c s (where c and c s are the monomers and excess salt concentration, respectively) is the concentration of all free monovalent ions, and b=10.2 Å (for HA) is the distance between charges along the chain. The case of HA is interesting because L 0 (cf helical structure) is not too large (contrary to DNA with L 0 ~500 Å), such that L e is more than a small perturbation; also all quantities still lie within the accessible size range of usual SANS.

In this letter, we look at hyaluronan shape and interactions when embedded in a mixture with another negatively charged polyelectrolyte chain, flexible, using Small-Angle salt NaCl (0, 10 -3 , 8×10 -3 , and 0.2 M) was progressively added to increase the solution ionic strength. SANS measurements were carried out at T=20 °C on spectrometers D11 (ILL, Grenoble) and PACE (LLB, Saclay) with configurations allowing a large range of q. The final spectra are given in absolute units of cross section (cm -1 ) following the standard procedures [7]. Also, the incoherent background of polyelectrolytes was subtracted. In dilute and high ionic strength solutions, it is classical to assume that the scattering is arising from isolated chains with I(q)(cm -1 )~() 2 V p P(q), where () 2 is the contrast factor,  the monomer volume fraction, and V p the chain volume.

To interpret the scattering, it is very useful to have a correct expression of the scattering of an individual chain (form factor), P(q). We use an expression [11] based on the expressions derived by Burchard and Kajiwara for rodlike structures [12] in which the form factor calculated by Sharp and Bloomfield [13] for finite wormlike chains of contour length (2) with x=L c L T q 2 /3 and valid for L c >4L T . Values of the empirical parameters =5.53 and =5.33, which contribute to the balance between the low-q and large-q terms, have been optimized in reference 11. To cope with the rather large index of polydispersity of HA (1.3), the contour length was taken as a fitting parameter. Eq 2 does not account for excluded volume interactions, which are negligible in the SANS q-range where L T is determined [11].

L c is used at low q.                                                                                            T c T c T c T qL qL q L L qL L L x x x x x x q P 2 exp
First consider simple HA solutions (without NaPSS-d): Figure 1 displays the scattering patterns (low-q light scattering is also presented after rescaling to SANS by the ratio between the contrasts of the two techniques [7]) for two salinities. At high I ([NaCl]=0.2 M,  -1 =6.8 Å ), the scattering curve for a polymer concentration c=1.8 g/l, that is below the overlap concentration, exhibits three domains: (i) a low-q smooth variation with measurable zero-q limit I(q=0), analogous to a Guinier regime, leading respectively to a radius of gyration equal to 350 Å and to a weight-average molecular weight, M w = 160K; (ii) an intermediate polymer coil regime in which the q dependence of the scattered intensity can be described by a power law with exponent close to -2, like in Gaussian coils, and finally (iii) a q -1 domain at higher q characteristic of a rigid rodlike behaviour for distances smaller than L T . This ensemble of variations can be considered as a form factor and fitted satisfactorily by the wormlike chain model with no excluded volume interactions (Fig. 1), yielding L T =43 Å (approximately equal to the intrinsic one) and L c =3880 Å corresponding to the theoretical value of a single 160K HA chain. Ionic strength screens chain interactions. FIG. 1. Scattering for simple 160K HA solutions at c=1.8 g/l. For clarity, the curve at 0M NaCl ( -1 =64 Å) was shifted by a factor 10.

At low I ([NaCl]=0 M,  -1 =64 Å), screening is weak. What is expected is that the absence of screening induces active repulsions between chains, which should lead to a privileged distance, in other words to a maximum in the scattering, the so-called "polyelectrolyte peak". This is not observed: instead, we see now, for decreasing q, a transition from a q -1 to a q -3 dependence (instead of q -1 to q -2 at high I). This steep upturn q (Å -1 ) HA probably overlaps and hides the polyelectrolyte peak (which is usually weak for some other natural polymers). The strong q -3 scattering is usually attributed to chain aggregates. This is confirmed by the low-q light scattering, which yields, through a Guinier law, a very large radius of gyration of 1040 Å and a molecular mass of 2.4×10 6 g/mol, around 15 times the mass of a single HA chain. Finally, dynamic light scattering experiments performed on the same solutions also confirm the presence of aggregates: they display the characteristic slow polyelectrolyte mode [7] usually observed for flexible polyelectrolytes at low ionic strength [6]. Let us now look at the q -1 regime. First, at the largest q, it overlaps perfectly with the one measured at high I (overlap is not seen on Figure 1 due to the shift used for clarity): aggregate scattering thus appears as negligible in this regime. The scattering can thus be fitted by the expression of Burchard and Kajiwara [12], using the same mass per unit length than that for single HA chain, and the same contour length L c =3800 Å. The q -1 domain is more extended towards small q, meaning that the persistence length of HA has increased. However, the q -1 -q - 2 rod-to-coil transition is masked by the aggregate signal, and it is not possible to deduce L T directly. Note that we need strong screening to prevent aggregation:  -1 must be smaller than 30 Å (for which it is still observed).

In mixtures of HA with deuterated NaPSS (where we "see" only HA), the effect of ionic strength is profoundly different. Both [NaCl]=0.2 M and 0 M lead now to the same shape of the HA scattering. No low-q light scattering data are obtained since NaPSS-d gives non-negligible signal in light scattering and the isolated HA intensity is not available. In Figure 2 we have selected the example of 160K HA (c=1.8 g/l) with deuterated NaPSS (c=0.96 g/l). A crossover between the q -2 coil regime (instead of q -3 ) and the q -1 rod-like regime is observed even for very low ionic strength solutions (i.e., for  -1 as large as 66 Å).

So, surprisingly, even at low ionic strength the low-q aggregation behaviour is not observed in mixtures. We are thus tempted to fit the scattering to the wormlike chain form factor Eq. 2: for the mixture at [NaCl] = 0 M (Fig. 2), we get a mass of 160K, and L c =4000 Å, just like for single chain: the scattering is identical to the form factor. We thus access to L T at low I, with a value of 71.1 Å. For the same mixture with added salt (high I), we obtain the same M w and L c , and L T =43.8 Å, i.e., values for a single neutral HA chain. Fits for all polyelectrolyte and salt concentrations investigated, and two molar masses, lead to:

M=16020K and L c = 4000200 Å, for 160K HA-NaPSS-d mixtures, and M=70070K and L c =175002000 Å, for 700K HA-NaPSS-d mixtures. The q -1 parts are superimposed for all conditions with only a weak dependence on excluded volume interactions and polydispersity in this q-range. This is fully similar to single HA chains behaviours, but for a much larger range of I (6.7 -1 66 Å) and is the main new fact of this report.

q (Å -1 ) This fact has an important consequence: now data sustain validity for extracting the electrostatic contribution, L e , and comparing its dependence over ionic strength with theory.

To get L e , we need to subtract from L T the value of L 0 : Since L 0 is not the focus of the paper, we get its value by subtracting from L T measured at high I ([NaCl]=0.2 M,  -1 =6.8 Å), the electrostatic contribution at this I as predicted by OSF theory (Eq. 1), L e =0.8 Å: the real value is bound to be as small anyway. The result, L e = L T -L T-0.2M + 0.8, is shown in Figure 3 as a function of  -1 . We observe that data lies upon to the line from OSF Eq. 1 without adjusting factor. This brings the second important result to this work, since to our knowledge; it is the first check of OSF theory with semiflexible polyelectrolyte covalent chains through direct measurement of their conformation. Former direct SANS measurements concerned flexible chains, NaPSS, and lead to a variation of L e closer to  -1 [14]. We previously attempted to follow such variation in hyaluronan only solutions, but had only indirect access to L e at low I because of aggregates scattering [7].

In summary, when HA semi-rigid chains are mixed with flexible polyelectrolytes bearing charges of the same sign at low ionic strength, an unambiguous set of experimental results shows that: (i) we neither observe an aggregation behaviour (ii) nor a polyelectrolyte peak; (iii) instead we observe a crossover between a q -2 and a q -1 regime, and finally (iv) a L e ~-2 variation. These two findings support each other through two lines of view: (i) the well known theory of Odijk finds here a direct check. (ii) The experimental knowledge of charged chain solutions and mixtures is enriched: aggregation and interactions, generally observed for single species, vanish here when a second charged chain of same sign is added.

FIG. 3. Dependence on  -1 of the HA electrostatic persistence length in the different mixtures.

The red line represents the actual OSF equation (Eq 1) without adjusting factor.

The origin of such aggregates in low ionic strength polyelectrolyte solutions (or, in terms of dynamic scattering, of slow modes) is not well understood. Since it occurs when long range electrostatic interactions are possible (low I), it has been proposed that polyions and counterions create a co-organized structure [8]. It occurs not only for HA only solutions as shown here, but also for flexible chains like NaPSS, which has been studied in detail [6]. But  -1 (Å) and thus induces counterion condensation as accounted by Manning theory [15]. For HA, b=10.2 Å, a much larger value, which does not induce condensation. Finally, non-electrostatic interaction between different species may play a role. Altogether, the situation is very different from a single species solution. The sharing of condensed counterions between two different species of polyelectrolytes characterized by strongly different structural parameters is difficult. It is thus possible that attractive forces between polyions mediated by this sharing become negligible, so that mixed aggregates cannot be stabilized. In other words, adding a different species creates a kind of "entropic", or "chaotropic screening". Note that mechanisms for regulating the colloidal stability have been proposed for other electrostatics systems [16]: negligibly charged colloidal microspheres, which flocculate when suspended alone in solution, undergo a stabilizing transition upon the addition of highly charged nanoparticle species that segregate to regions near microspheres because of their repulsive electrostatic interactions. In the present case the situation is different: in particular, both polyelectrolytes are well charged, and the length scales are quite shorter. Beyond absence of aggregation, we have to account for absence of polyelectrolyte peak or any interchain HA correlation (the zero q limit stays proportional to the single chain molar mass): this also could be linked with larger entropy in our mixture.

*Corresponding authors. Email address: eric.buhler@univ-paris-diderot.fr; schweins@ill.eu

[1] J.F. Joanny and J.L. Barrat, in Advances in Chemical Physics (Wiley, New York, 1996), Vol. XCIV.

FIG. 2 .

 2 FIG.2. Scattering for [160K HA (1.8 g/l)-80K NaPSS-d (0.96 g/l)] mixtures at low ionic strength I ( -1 =55 Å, for clarity the curve has been shifted by a factor 10) and high I ( -1 =6.8

2 the

 2 two species have strongly different parameters: (i) NaPSS has a short intrinsic persistence length, L 0,PSSNa =9.5 Å[14], whereas, for HA, L 0  50 Å. (ii) The distance between charges along the chain is b = 2.45 Å for NaPSS, which is much shorter than the Bjerrum length l B


Jacques Jestin

Fabrice Cousin

Isabelle Dubois

Christine Ménager

Ralph Schweins

Julian Oberdisse

François Boué

Ralf Schweins

ANISOTROPIC REINFORCEMENT OF NANOCOMPOSITES TUNED BY MAGNETIC ORIENTATION OF THE FILLER NETWORK

published or not. The documents may come

Introduction

Progress in both design of innovative materials and understanding of their properties has been achieved in the past two decades by extending polymer reinforcement by fillers to nanofillers 1 . Among the still open challenges, the improvement and possibly tuning of a specific material property through a simple external trigger is of particular importance. A promising pathway is to bring in a new degree of freedom, e.g., by using magnetic, instead of classical inorganic particles as filler. The design of a functional nanocomposite material with the possibility to orient the filler inside the matrix should allow to tune macroscopic material properties, and in particular the mechanical ones, using a simple external magnetic field. This concept has been explored in the past twenty years by many research teams, mostly studying the effect of micron-size particles or fibres (needles) [2][3][4][5] . Two routes are possible: applying the field on the material after synthesis or applying it during the synthesis. We use here this second approach for nanoparticles; in such case, the question of the mechanical consequences of local anisotropy due to specific orientation of a nanofiller, i.e. particles with a size comparable to polymer network mesh sizes, has still to be solved.

Here we present a new material based on a dispersion of magnetic nano-particles in a polymer matrix. This is achieved in a controlled way by adapting the knowledge on these systems in solution ("Ferrofluids") 6 to our own method of dispersion of silica nano-particles in a polymer matrix made of latex beads [7][8][9] : via solvent (water) casting of a suspension of magnetic nanoparticles and polymer latex, a "Ferrolatex nano-composite" is formed. In this article, we will show that achieving these steps under the action of a weak magnetic field induces orientation responsible of a strong anisotropy of the reinforcement. Moreover, this mechanical anisotropy can be understood by measuring the spatial arrangement and orientation of the particles on a scale between one nanometer and two hundred nanometers, using Small Angle Neutron Scattering (SANS).

Sample preparation

The synthesis of a composite made of a latex film filled with magnetic particles requires two main steps (fig. 1). The first main step is the synthesis of the magnetic particles according to a well known process described in the Methods Section. In a second time, in the same batch, the particles are covered with a thin layer of silica. This silica shell makes the particles pH compatible with the latex beads while keeping the initial size and the magnetic properties of the core. Hence we can use the technique developed for introducing silica particles in a latex film 7,9 . The colloidal suspension of particles is mixed with a polymer bead (R200 Å) suspension at a fixed, high pH, followed by evaporation of the (aqueous) solvent. The latex nanospheres come in contact, and the capillary forces compress them into adjacent polyhedrons. We know 7 that under deformation above Tg, such matrix deforms affinely (homothetically) to the macroscopic deformation, due to the elasticity of the membrane.

The colloidal mixture, with a proportion of magnetic nano-particles to the latex spheres of 5% by volume is poured into rectangular 160.1 cm 3 moulds and dried (at T  60°C), i.e. above the Film Formation Temperature which is itself close to the glass temperature transition of the latex (Tg  33°C).

During evaporation, a weak constant field (150 gauss) is applied in order to orient the magnetic filler. A similar sample is also evaporated in the same mould without magnetic field.

We call it the "zero-field cast" sample.

Mechanical reinforcement

We have performed mechanical tests with a home-made uniaxial elongation machine.

Keeping in mind the direction of the magnetic field during casting, we have the opportunity to study two simple but fundamental cases, stretching parallel or perpendicular to the field, using two rectangular films (1x6x40 mm 3 ) cut from the same sample. This allows a direct comparison of the stress-strain isotherms in the two directions. The samples are first immersed in a silicone oil bath at a temperature T g + 27°C, then, elongated at constant velocity gradient. When the desired elongation is reached, namely =1.5, 2 and 3, the samples are immediately frozen by immersion in a carbonic ice bath. The reference case for the mechanical tests is given by the stress-strain isotherm of the pure latex matrix. It is used as a normalisation, i.e. we divide by all the stress-strain isotherms measured for the nanocomposite: this gives us a reinforcement factor R () for each composite with respect to its own matrix. To check the reproducibility of the synthesis of the magnetic nanoparticles, we have pulled a second series of films synthesized out of a second batch of magnetic nanoparticles with similar sizes. The four stretching curves  versus  are plotted on Fig. 2a together with the ones of the pure matrix and of the zero-field cast film. For all samples with nanoparticles, the shape of the curves is similar: compared to the pure latex, they show a fast increase at the beginning of the elongation, while the shape at large  tends to be similar.

There is a very strong effect for the zero-field cast sample, whereas after field-casting we can observe that the reinforcement sample is lower by one order of magnitude.

But the striking effect is the difference between the field-cast samples stretched in parallel and perpendicularly to the magnetic field. They show a very different behaviour, especially at low deformation. Moreover, the mechanical tests for the two batches of magnetic nano-particles show a very nice reproducibility for the two directions.

If we plot in Fig. 2b the reinforcement factor R () as function of the deformation , we see a strong reinforcement for the small deformation and a rapid decrease of R () with  toward a constant value, noticeably smaller (but larger than one). This is very similar to what formerly described for nanolatex composites filled with silica nanoparticles 9 .The curve aspect is common to the two directions. The important fact is that the reinforcement factors obtained are much higher when the field is along the deformation direction than when the field is perpendicular to it. We quantify the anisotropy of the mechanical properties of a given sample by the ratio of the parallel reinforcement to the perpendicular reinforcement (Fig. 2c).

It is more than a factor two. It has also to be noted for the discussion below that such anisotropy in R () vanishes for large elongations ( > 2).

In summary, we are able to obtain a strong effect of the mechanical properties of the nanocomposites under orientation by the field. The question is now to try to correlate the macroscopic effect with the local organization of the filler network in the matrix and its evolution under the mechanical strain. This is reported in the following using SANS measurements analysis.

Local structure

SANS measurements have been performed on samples after each successive step of the synthesis. In the following, figures from 3 to 5 will be presented in the same way: above 2D intensity maps, in the middle the radially averaged intensities as a function of the scattering vector, and at the bottom a sketch in the real space illustrating the structure of the network of aggregates. First, Fig. 3a shows results for magnetic particles in dilute aqueous solution at low volume fraction ( = 0.001) before and after the silica-covering process. The scattering is obviously isotropic and its radial-average can be fitted at large q to the one of spherical primary particles with a log-normal polydisperse distribution of average radius 35 Å and variance  = 0.3. The low q rod-like scattering (q -Df , fractal dimension D f = 1) suggests that they are organized in small "primary clusters". The mean number of particles N prim in a cluster is of the order of 3 to 4 particles, as obtained from the fit to the scattering of a fractal aggregate of D f = 1 (cf. ref. 10 for details). Second , patterns after casting and before stretching, are presented in Fig. 3b. As expected, the zero-field cast sample is isotropic. The radial average displays the characteristic signature of the formation of aggregates during film drying. Fortunately, the size of such large objects is observable in our q-window, as illustrated by a plateau in the scattering curve at low q. This intensity limit corresponds to an aggregation number of around 10 primary clusters (~ 30 nanoparticles). At slightly larger q, where the internal structure of the aggregates is probed, we observe a q -2 behaviour, i.e. a fractal dimension D f  2, as typically encountered for either Diffusion Limited Aggregation 11 (DLA, D f = 1.8 in 3D) or Reaction Limited Aggregation 12 (RLA, D f = 2.1 in 3D). At larger values of q (around 4.10 -2 Å -1 ), we see an oscillation in the scattering curve which corresponds to the correlations between primary clusters inside the aggregates. Let us now come to the field-cast film: it gives a nicely anisotropic scattering pattern (Fig. 3c). The elliptic shape with large horizontal axis, i.e. transverse to the field, is characteristic of objects elongated vertically: thus there are aggregates aligned parallel to the field. To quantify this effect, let us radial average the scattering inside an angular sector along one direction, either vertical or horizontal, with a width of 25°. For small q, the intensity is smaller along the vertical axis than along the horizontal one. The aggregates are aligned on a larger scale along the field (vertical here) and we can assume that they form "chains of aggregates" inside the matrix. The intensity along the horizontal axis shows a strong q -3 decrease. This exponent 3 is mostly encountered for compact objects, composed here of chains of aggregates that we will define as "supra-aggregates". The scattering is still increasing at the lowest q of the experimental window. This means that the size of the supra-aggregates is too large to be probed here. The sizes of the structural organization of the aggregates inside one supraaggregate are also too large to be accessible within the experimental q-range. For the high-q values, the intensities along the vertical and the horizontal axis are superimposed with the zero-field sample. This indicates that the local structure of the aggregates, composed of the primary clusters, is not affected by the application of the magnetic field. To summarise this section, we have succeeded in orienting the filler as chains of aggregates, inside the nanolatex composite using a constant field.

Let us now discuss the evolution of the structure of the filler network under stretching. We start with the first case, where to the elongation (vertical) is parallel to the field Figure 4 shows the 2D intensity map for each . When the elongation rate increases, the anisotropy is increasing towards a surprising clear cut hexagonal form. This hexagonal form is due to the rise of a maximum in the vertical direction. This maximum appears at a small q value for the first elongation ( = 1.5) and its position moves to higher q values at higher elongations. This behaviour is highlighted in the S(q).q² versus q representation of Fig. 4a and4b, convenient here since the scattering decays like q -2 at intermediate q. S(q) is the result of the division of the I(q) by the form factor P(q) of the primary clusters. The data show that elongation of the sample induces organization of the aggregates with a characteristic distance between the aggregates in the vertical direction. Please note that the curve for the unstretched sample showed no maximum in the available q range. Since the correlation peak is shifted towards higher q when the elongation increases, the characteristic distance decreases. Intuitively, one would expect the contrary, as the aggregates move away from each other along the stretching direction. This implies that under strong deformation some aggregates coming from neighbouring chainlike supra-aggregates intercalate vertically between two aggregates. Such a mechanism results in an effective decrease of the vertical inter-aggregate distance, as illustrated in the real space sketches of Figure 4. Along the horizontal direction, the intensity decreases compared to the isotropic signal: the supra-aggregates are brought closer due to the lateral shrinking of the sample which increases the effective compressibility between them.

In Fig. 5, we present the second case where the stretching direction (here horizontal) is perpendicular to the chainlike supra-aggregates. In addition to the isotropic intensity map, two elongation ratios are presented, =1.5 and =2. The result of the deformation is completely different in this case compared to the previous one; the scattering pattern becomes isotropic as the elongation is increased. Here again this is highlighted when one plots S(q).q² as function of q along both vertical and horizontal directions of the 2D intensity map: the anisotropy is important for the non-stretched sample (Fig. 5a) and becomes small for the elongated one (Fig. 5b). The orientation of filler breaks under the mechanical constraint: the aggregates are reoriented isotropically. Moreover, the supra-aggregates are destroyed: the final scattering of sample stretched to = 2 superimposes nicely to the scattering from the zero-field cast sample (black symbols).

Finally, when confronting the structural organization of the filler network deduced from SANS to the reinforcement, a consistent picture emerges. Under stretching, the parallel oriented chains rearrange along the deformation direction due to a contraction of the interconnecting particle network. In contrary, the perpendicularly oriented network breaks under deformation. This leads to a higher reinforcement factor in parallel direction. In the perpendicular direction, the final structure of the chains is resumed to the organization of the zero-field cast sample (at least in the available q range), but the latter has a higher reinforcement factor. We propose to attribute this difference to the existence of larger aggregates connected at larger scales.

Conclusion

To summarize, we have designed an original material which is structured on the nanoscale and which displays anisotropic and tuneable mechanical properties. The samples have been formulated rather easily by mixing aqueous suspensions of polymer and magnetic colloids, followed by casting. These nanocomposites bear magnetic properties, and therefore the possibility of pre-orientation when cast under field. Detailed insight on dispersion by Small Angle Neutron Scattering shows that primary particle aggregates are organized at the nanometer scale, and align in the presence of a field. Such anisotropy has a clear effect on the mechanical reinforcement brought by the fillers. The interesting novelty is the anisotropic reinforcement: it is larger by a factor 2 at low deformation along the field than transverse to it.

Microscopically, this can be understood with the SANS-data: while strain perpendicular to the chains destroys chain ordering, strain parallel to the field induces additional organisation.

Thus anisotropic mechanical reinforcement is obtained through anisotropic interconnection of nanoparticles, which is a one-dimensional version of the filler network effect. We note that all these effects vanish at large  (>2).

In addition to such controlled anisotropy of our nanocomposites, we would like to point out another contribution of this study, which concerns the conceptual side. Our system opens the way to discriminate between the two dominant contributions assigned to the filler in reinforcement mechanisms: the role of the particle interface and the one of the filler network.

The first one includes interaction between the particles and the polymer, and the consequences on the chains dynamic [13][14][15][16][17] ; the second one interconnected networks and/or aggregation 18 . Experimentally, it appears to be very difficult to separate the two effects, as one usually influences the other. With the new system, we are able to propose a simple alternative to act on the nanoparticles filler network without important modification of the interface. Indeed the simple orientation of the filler network does not involve modification of the matrix chains themselves (at variance with, for example, strain exerted on the full sample).

Methods

Ferro-particles synthesis:

The nanoparticles are made of maghemite (-Fe 2 O 3 ). They are chemically synthesized in water by coprecipitation of FeCl 2 and FeCl 3 in an alkaline solution 19 . A size-sorting procedure 20 made at the end of the synthesis enables to reduce the polydispersity of the nanoparticles. The final particle size is obtained by magnetization measurements and by SANS experiments that give the same results. The size distribution is described by a lognormal law with a mean radius R 0 of 35 Å and a polydispersity index  of 0.3. At the end of the synthesis the naked nanoparticles are stabilized at pH 12 by N(CH 3 ) 4 + counterions. In order to increase their stability range (their PZC is 7.2 and they are only stable for pH > 10), their surface is coated by a thin shell of silica. The silica-coating process is based on a twostep procedure method described by Philipse et al 21 to obtain silica particles with a magnetic core, that we limit here to the first step. A solution of SiO 2 , Na 2 O is added to a suspension of naked nanoparticles at pH 12 inducing the formation of silica oligomers in the solution. The oligomers adsorb on the nanoparticles surface. The concentrations of particles and silica correspond to a molar ratio [Fe]/[Si] = 1 and are chosen weak to prevent the formation of silica bridges between particles and thus irreversible aggregation. In order to eliminate silica oligomers in excess the suspension is dialyzed against a reservoir constituted by a NaOH solution at pH 10. The nanoparticles have the same size than before the silica-coating process (SANS measurements) but they are stable from pH 4 to pH 12. A very thin layer of silica has thus been coated on the particle that modifies their surface properties but not their magnetic properties.

Composite nano-latex:

The nanolatex was kindly provided by Rhodia. It is a solution of core-shell latex of poly(methyl methalycrate) (PMMA) and poly(butylacrylate) (PBuA), with a hydrophilic shell containing methacrylic acid that enables colloidal stability in water. The approximate size given by Rhodia is R ~ 200 Å. Before mixing it is dialyzed against the same reservoir as the one used for the dialysis of the silica-coated magnetic nano-particle to impose the same pH and ionic strength to the two components of the mixture.

The two solutions are then mixed with  nanoparticles / nanolatex .= 0.05. The final mixture is degassed under primary vacuum about 1 day to avoid bubble formation. The solvent is then slowly evaporated during 4 days at T = 60°C under atmospheric pressure to get a homogeneous bubble-free film. During the drying step a magnetic field of 150 Gauss is imposed to the sample in the plane of the final film.

Stress-strain isotherms and the reinforcement factor:

Samples for stress-strain isotherms are brought to constant thickness using sandpaper. They are stretched up to rupture in a controlled constant-rate deformation ( = 0.0016 s -1 ), at T = 60°C, i.e. well above the glass transition temperature of the matrix (nanolatex Tg = 33°C).

The force F(), where  = L/L 0 defines the elongation with respect to the initial length L 0 , is measured with a HBM Q11 force transducer, and converted to (real) stress inside the material . The deformation of the film is assumed homogeneous, and incompressible. It is recalled that in our silica-latex system, the physico-chemical parameters that control the morphology of aggregates of silica nanoparticles are the silica concentration, the pH and the salinity in solution before solvent evaporation. We have shown previously that the rheological properties of the pure nano-latex matrix are pH-dependent 10 . This is one of the reasons why we analyze our data in terms of the nano-composite reinforcement factor R (λ) = ()/ latex (), where the stress of the pure matrix  latex () has been measured at the same pH.

Small Angle Neutron Scattering:

Experiments have been performed at LLB (Saclay, France) on beam line PAXY, at ILL (Grenoble, France) on beam line D11 and at HMI (Berlin, Germany) on beam line V4. On PAXY, a set of two sample-to-detector distances 1 m and 5 m at wavelength 6Å give us a q range between 8.10 -3 et 0.3 Å -1 . On D11, a set of two wavelengths, 6 and 12 Å, with four sample-to-detector distances, 1m, 5m 20m and 34m, gives us access to a q range comprised between 8.10 -4 and 0.3 Å -1 . On V4, a set of three sample-to-detector distances 1m, 4m and 12m and two wavelengths, 6 and 12 Å, gives us a q range from 2.10 -3 to 0.1 Å -1 . Data treatments have been done following standard procedures, with H 2 O as calibration standard, to get absolute intensities. Incoherent scattering and background contributions of the nanolatex films have been subtracted from the pure latex matrix scattering (the incoherent scattering from maghemite nanoparticles is null). In the simplest case of centro-symmetrical mono-disperse particles, the scattered intensity I(q) can be written as function of the wave vector q like I(q)= ² V P(q) S(q),  is the volume fraction of the filler,  the difference of scattering length density of particles with the one of the matrix or the solvent, V the volume of the particle, P(q) the form factor and S(q) the structure factor. For dilute systems, the structure factor is close to 1 because interactions are negligible. For concentrated systems with repulsive interactions, S (q) presents a correlation peak at q*=2/D where D is the average distance between the scattering mass centres. For attractive systems, aggregates of initial particles can be formed with different compactness. For open systems, one gets a fractal organization of particles with a fractal dimension D f and the scattering spectra decay like q -Df whereas for dense systems high compactness can be obtained due to close packing volume fraction.

Figure 1

 1 Figure 1 Sketch illustrating the route for incorporating magnetic nanoparticles in polymer films by latex film formation and the orientation of the fillers with a constant field.

Figure 2 a

 2 Figure 2 a, Stress-strain isotherm for: pure latex matrix, zero-field composite sample containing 5% in volume fraction of magnetic nanoparticles, and nano-latex films cast under constant field and stretched in directions parallel and perpendicular to the field. b, Reinforcement factor as function of the deformation for the field-cast samples under constant field. c, parallel on perpendicular ratio of reinforcements for the field-cast samples under constant field.

Figure 3

 3 Figure 3 SANS scattering of the samples at rest. From up to down: 2D spectra at the lowest q-range probed in the experiment DONNER DISTANCE ET LONGUEUR D ONDE ?; 1D scattered intensity; corresponding scheme of the structure. a, the initial aggregates of magnetic particles covered with silica layer, the full red line to the form factor of an isolated nanoparticle and the full blue line correspond to a cluster of 3-4 nanoparticles with D f = 1 b, the zero-field composite sample and c, the nano-latex composite film evaporated under field.

Figure 4

 4 Figure42D scattering of the stretched nano-latex film when the elongation direction is along the field-oriented fillers. Radial grouping of the S(q).q² versus q along the vertical a, and the horizontal direction of the picture b, for each elongation values and sketch of the structure in the real space. The hexagonal aspect of the 2D picture is due to the apparition of a correlation peak which corresponds to a characteristic distance between the aggregates along the stretching direction (black arrow for =1.5). The green arrow for =3 signals a smaller

Figure 5

 5 Figure 5 SANS scattering of the stretched nano-latex film when the elongation direction is perpendicular to the field-oriented fillers. Radial intensities grouped along the vertical and the horizontal direction of the 2D picture as function of q. a, sample before stretching, b,

Acknowledgements

We acknowledge the important help of Mireille Nuyadge (undergraduate trainee at LLB & LI2C) and Delphine Talbot (LI2C) during synthesis of ferrofluids (LI2C), and of Jean Chevalet (LI2C) for design of the film casting device under field.


Cecile Barron

Saulnier

Cécile Barron

Christine Bar-L'helgouac'h B

Martine Champ

Luc Saulnier
email: luc.saulnier@inra.fr

predictors of the dietary fibre content and their nutritional properties in wheat products

Keywords: bread, biscuits, pasta, dietary fibre, enzymatic hydrolysis, xylanase

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

Introduction

Epidemiological studies have shown that high dietary fibre (DF) diets and whole grain consumption are associated with diminished risk of coronary heart disease, colon cancers, inflammatory bowel disease, and metabolic syndrome [START_REF] Stephen | Dietary fibre in Europe: current state of knowledge on definitions, sources, recommendations, intakes and relationships to health[END_REF]. Thus, health authorities around the world recommend that adults consume 25 g to 35 g of fibre per day [START_REF] Stephen | Dietary fibre in Europe: current state of knowledge on definitions, sources, recommendations, intakes and relationships to health[END_REF]. Bread, pasta and non-bread product such as wheat flour tortillas are staple foods in many countries and wheat flour is the main component of many food products such as pizzas, biscuits and cakes. Obviously, an increase in the DF content of these wheat-based foods might help to better meet dietary recommendations for DF intake. However, DF are a complex mixture of chemically heterogeneous carbohydrate components, and the quantity and nature of DF in wheat-based foods vary, in particular, depending on the "refining" of the flours (white flour vs whole grain flour) and the food manufacturing processes that can affect for example the amount of resistant starch. Although some of the physicochemical properties of DF (solubility, viscosity, bulking) are clearly related to important nutritional outcome [START_REF] Mcrorie | Understanding the Physics of Functional Fibers in the Gastrointestinal Tract: An Evidence-Based Approach to Resolving Enduring Misconceptions about Insoluble and Soluble Fiber[END_REF], the mechanisms by which the consumption of DF modulates health are multiple and not fully elucidated. However, their role in maintaining diversity and stability of the microbiota probably plays a decisive role in the regulation of the immune and metabolic systems and therefore on health [START_REF] Sonnenburg | Diet-microbiota interactions as moderators of human metabolism[END_REF]. In this respect, a key feature of DF is their fermentation pattern which is related to their chemical structure and to the ability of microorganisms to produce the glycosyl-hydrolases allowing their degradation in the gut [START_REF] Flint | Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis[END_REF].

As previously stated, DF are a complex mixture of chemically heterogeneous carbohydrate components and their measurements have been based on sequential enzymatic-gravimetric assays mimicking human digestion. These methods have undergone numerous evolutions [START_REF] Macagnan | Dietary fibre: The scientific search for an ideal definition and methodology of analysis, and its physiological importance as a carrier of bioactive compounds[END_REF] but the AOAC method 985.29 [START_REF] Prosky | Determination of insoluble, soluble, and total dietary fibre in foods and food products[END_REF] was widely used for the determination of total dietary fibre (TDF) in foods. Prosky HMwSDF. An evolution of the AOAC 2009.01 method [START_REF] Mccleary | Modification to AOAC Official Methods 2009.01 and 2011.25 to Allow for Minor Overestimation of Low Molecular Weight Soluble Dietary Fiber in Samples Containing Starch[END_REF] was introduced to correct an overestimation of the LMwSDF fraction in starch-rich foods and particularly in cereal-based foods [START_REF] Brunt | Improvement of the AOAC 2009.01 total dietary fibre method for bread and other high starch containing matrices[END_REF][START_REF] Mccleary | Determination of total dietary fibre and available carbohydrates: A rapid integrated procedure that simulates in vivo digestion[END_REF]. Despite their successive improvements, these methods made of several steps are time-consuming and results obtained are still under debate [START_REF] Hell | Analytical techniques for the elucidation of wheat bran constituents and their structural features with emphasis on dietary fiber -A review[END_REF]. Moreover they do not take into account the chemical and physico-chemical diversities of DF that is far more complex than proposed by a classification in HMwDF/LMwSDF and insoluble/soluble fractions [START_REF] Gidley | Functional categorisation of dietary fibre in foods: Beyond 'soluble' vs 'insoluble[END_REF].

In view of the diversity of DF and of their mechanisms of action, reliable method to quantify their amount is crucial to characterize cereal products. Such method could help to monitor the development of DF enriched wheat-based foods and could also help to better assess some of their important nutritional property such as their fermentation ability. The wheat grain contains about 12-14% of DF [START_REF] Gebruers | Variation in the Content of Dietary Fiber and Components Thereof in Wheats in the HEALTHGRAIN Diversity Screen[END_REF] but this complex organ is composed of different tissues composed of cell wall polysaccharides exhibiting various properties and composition [START_REF] Barron | Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition[END_REF][START_REF] Saulnier | Cell wall deposition and metabolism in wheat grain[END_REF]. In wheat grain, arabinoxylans (AX) are the major polymers in the cell wall and therefore a major component of DF. In the starchy endosperm cell walls represent 3 to 5% of the tissue and are essentially constituted by AX with a lower proportion of (1,3)(1,4)-linked beta-d-glucans [START_REF] Saulnier | Cell wall deposition and metabolism in wheat grain[END_REF] and cellulose [START_REF] Gartaula | Quantitative structural organisation model for wheat endosperm cell walls: Cellulose as an important constituent[END_REF]. In this tissue, AX are partly water soluble and exhibit an arabinose to xylose ratio (A/X) of about 0.6. In the most outer layers cell wall polysaccharides represent up to 50% of the tissue and are mainly composed of cellulose and complex AX that are mostly water insoluble and exhibit a structure more heavily substituted by arabinose than in starchy endosperm (A/X close to 1) [START_REF] Antoine | Individual Contribution of Grain Outer Layers and Their Cell Wall Structure to the Mechanical Properties of Wheat Bran[END_REF][START_REF] Barron | Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition[END_REF][START_REF] Parker | The phenolic acid and polysaccharide composition of cell walls of bran layers of mature wheat ( Triticum aestivum L . cv . Avalon ) grains[END_REF]. In addition, hydroxycinnamic acids that are ester-linked to some of the arabinose side-chains of AX [START_REF] Harris | The distribution of ester-linked ferulic acid in the cell walls of angiosperms[END_REF] play an important role for polymer interactions and cell wall properties, as they can dimerize under oxidizing conditions and act as bridging agent interconnecting AX chains together and with lignins [START_REF] De Oliveira | Ferulic acid: A key component in grass lignocellulose recalcitrance to hydrolysis[END_REF]. The susceptibility to enzyme attack is clearly related to AX structure [START_REF] Bonnin | Behaviour of family 10 and 11 xylanases towards arabinoxylans with varying structure[END_REF] and is deeply impacted by the interactions of AX within the cell wall. Outer tissues of the grain (pericarp) are not degraded by pure microbial xylanases [START_REF] Beaugrand | Arabinoxylan and hydroxycinnamate content of wheat bran in relation to endoxylanase susceptibility[END_REF][START_REF] Ordaz-Ortiz | Classification of Wheat Varieties Based on Structural Features of Arabinoxylans As Revealed by Endoxylanase Treatment of Flour and Grain[END_REF], whereas AX from starchy endosperm or aleurone layer are extensively hydrolysed by these enzymes [START_REF] Ordaz-Ortiz | Classification of Wheat Varieties Based on Structural Features of Arabinoxylans As Revealed by Endoxylanase Treatment of Flour and Grain[END_REF]. The resistance is mainly explained by the covalent interactions through ferulic acid bridges between AX chains and with lignin. These interconnections are much more numerous in the outer pericarp that is resistant to enzyme attack than in cell walls of endosperm tissues (including aleurone layer) [START_REF] Chateigner-Boutin | Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development[END_REF][START_REF] Saulnier | Cell wall deposition and metabolism in wheat grain[END_REF] that are degraded by microbial xylanases. Actually, the ability to be degraded by microbial enzymes (e.g. microbiota) differs largely between isolated polymers and polymers as present in the cell wall of the different tissues [START_REF] Rose | Structural differences among alkali-soluble arabinoxylans from Maize (Zea mays), Rice (Oryza sativa), and Wheat (Triticum aestivum) brans influence human fecal fermentation profiles[END_REF]. The susceptibility to enzyme degradation of the different tissues of the grain has potentially important health impact as isolated AX polymers and arabino-xylo-oligosaccharides (AXOS) have been shown to favour the development of beneficial bacteria [START_REF] Broekaert | Prebiotic and Other Health-Related Effects of Cereal-Derived Arabinoxylans, Arabinoxylan-Oligosaccharides, and Xylooligosaccharides[END_REF][START_REF] Neyrinck | Prebiotic effects of wheat Arabinoxylan related to the increase in bifidobacteria, roseburia and bacteroides/prevotella in diet-induced obese mice[END_REF] with an increase in Bifidobacteria/Lactobacilli as well as in Bacteroides/Prevotella and Roseburia groups [START_REF] Despres | Xylan degradation by the human gut Bacteroides xylanisolvens XB1AT involves two distinct gene clusters that are linked at the transcriptional level[END_REF] In this work, the tissue composition of different millstreams has been measured [START_REF] Hemery | Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions[END_REF]) and compared to their total DF amount measured with the AOAC 2009-01 method.

Assuming that the most peripheral tissues are mainly constituted of DF, the tissue composition could be used to estimate the total DF content in millstreams. However, such approach could not be extended to wheat-based foods. Therefore, knowing that AX are the main components of cell walls in the different tissue of the wheat grain, biochemical determination of AX content has been investigated as a possible predictor of DF content. In addition, adding an enzymatic hydrolysis step with a xylanase and quantifying the amount of AX released was carried out in order to evaluate the potential fermentation capacity of various wheat millstreams.

Material and Methods

Wheat grains and milling fractions

Different millstreams were produced from bread wheat (Caphorn cv; Triticum aestivum L.) and durum wheat (Miradoux cv; Triticum durum Desf). Fractions were obtained from conventional milling technology with specific diagrams to bread wheat in ENILIA-ENSMIC pilot mill (Surgères, France) or durum wheat in UMR IATE pilot plant (Montpellier, France). In addition, a PeriTech debranner (Satake, UK) was used to obtain debranning fractions differing in their extraction rate.

Two specific fractions named F -and F + were also produced from Caphorn grains. Fraction F - corresponded to a debranning level of 3.8% in weight of the grain, and fraction F + was subsequently isolated at a higher debranning level (recovered between 6.4% and 12.9% in weight of the grain). The other debranning fractions (A to J) from both bread and durum wheats were also produced with this equipment. Different debranning levels and successive debranning steps were used in order to obtain fractions differing in their outer tissues content.

Wheat-based foods

Bread, pasta and biscuit were produced with white flour (regular product) or with flour enriched in DF by incorporating F -and F + fractions.

Délifrance provided breads produced on an industrial line in Marquette-lez-Lille (France). Regular bread (R-bread) was made with white flour. The same flour that incorporated fraction F -(20.5% weight basis) or fraction F + (50.8% weight basis) was used for F --bread and F + -bread, respectively.

Precooked breads (55g) were used for analysis.

Mondelez provided biscuits produced on a pilot line at its R & D centre (Saclay, France). Regular biscuits (R-Biscuit) contained fat (15%), sugars (23%) and the rest of the formula was completed with cereals (2/3 wheat flour and 1/3 oat flakes) or incorporated fraction F -(10.5%) or fraction F + (27.1%) completed with cereals for F --biscuit and F + -biscuit, respectively. Pasta were provided by Panzani. Pasta was made from 100% durum wheat semolina for regular product (R-pasta) and incorporated fraction F -(9%) or fraction F + (25.9%) for F --pasta and F + -pasta, respectively.

DF content and biochemical characterization

DF content in the different samples was determined according to the AOAC 2009-01 method [START_REF] Mccleary | Determination of Total Dietary Fiber (CODEX Definition) by Enzymatic-Gravimetric Method and Liquid Chromatography: Collaborative Study[END_REF] on a dry matter basis. High molecular weight DF (soluble and insoluble fractions) are reported as HMwDF and low molecular weight soluble DF as LMwSDF, and TDF = HMwDF + LMwSDF. In addition, resistant starch was measured in wheat-based foods according to AOAC method 2002-02. In food, the relative maximum deviation between duplicates was 14%, 30% and 16% for HMwDF, LMwSDF and TDF, respectively. In wheat fractions coefficient of variation between duplicates was 2%, 7% and 3% for HMwDF, LMwSDF and TDF, respectively.

The AX content was calculated from neutral sugar analysis. Prior to neutral sugar analysis or enzymatic degradation, samples were ground in a Freezer/Mill 6700 cryogenic grinder (Spex, USA) then 0.8 g were treated with 5 mL of boiling 80% (w/v) aqueous EtOH for 10 min and the insoluble residue separated by centrifugation (6300 g, 10 min). The procedure was repeated once and the residue finally washed with 5 mL of 95% (v/v) EtOH and again separated by centrifugation (6300 g, 10 min). The supernatant was discarded and the residue dried in two steps: first in an oven at 40°C for 24 h and then in a vacuum-oven over P2O5 at 40°C for 24 h.

The neutral sugar composition of samples was identified, after acid hydrolysis, using gas chromatography of alditol acetates [START_REF] Englyst | Improved method for measurement of dietary fiber as non-starch polysaccharides in plant foods[END_REF]. Acid hydrolysis of samples was carried out as previously described [START_REF] Saulnier | Cell-Wall Polysaccharide Interactions in Maize Bran[END_REF]: the samples were pre-hydrolysed with 72% (w/w; 26 M) sulphuric acid for 30 min at 25°C and then hydrolysed into monomers at 100°C for 2 h in 1 M sulphuric acid. Analyses were performed in duplicate and AX content (AXtot) was calculated by summing the amount of arabinose and xylose. The relative maximum deviation between duplicates was 3% or less.

Samples (100 mg) were incubated in water (1 mL) with an endoxylanase (EC 3.2.1.8) from Trichoderma viride (Xylanase M1, Megazyme, Ireland) at 40°C for 16 h under continuous stirring.

Xylanase amount (2.4 U/mg of AX) was adjusted according to the amount of AX determined in the sample [START_REF] Ordaz-Ortiz | Classification of Wheat Varieties Based on Structural Features of Arabinoxylans As Revealed by Endoxylanase Treatment of Flour and Grain[END_REF]. The reaction mixture was centrifuged (6300 g, 10 min) and 0.7 mL of supernatant was removed and boiled for 10 min to inactivate the enzymes. Neutral sugar content in the supernatant was identified after acid hydrolysis (1 M H2SO4, 100°C, 2 h) using gas chromatography of alditol acetates [START_REF] Englyst | Improved method for measurement of dietary fiber as non-starch polysaccharides in plant foods[END_REF]. Analyses were performed in duplicate and AX amount released by the enzyme (AXenz) was calculated by summing the amount of arabinose and xylose in the supernatant and expressed as weight % of the starting sample. Coefficient of variations between duplicates were 3% or less.

Tissue composition

Tissue composition was assessed by the biochemical markers methodology as previously described [START_REF] Hemery | Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions[END_REF]. Reference values were obtained from the chemical analysis of pure dissected tissues except for starch amount in starchy endosperm, which was deduced from starch amount in high purity flour. For Miradoux cultivar, separation between the outer pericarp and the so-called "intermediate layer" could not be achieved by hand dissection. Therefore, both tissues were monitored at the same time by looking at the amount of alkylresorcinols.

Results & discussion

Determination of DF contents in wheat products

The TDF content was measured by the AOAC 2009-01 method and the amounts of HMwDF and LMwSDF are reported in Tables 1 and2. TDF contents were determined in wheat-based foods (pasta, biscuit, bread) made from white wheat flour and enriched in DF with specific wheat milling fractions (Fractions F+ and F-) and over a wide range of wheat milling fractions obtained by classical milling and by debranning. This set of samples provided a large range of TDF contents ranging from 3% up to 70% in milling fractions. Within wheat-based foods (pasta, biscuits and bread) this range was only 3-15%. The higher contents (TDF > 50%) were obtained for bran and peeling fractions from common wheat and durum wheat that were especially rich in HMwDF. For milling fractions the variation coefficient of HMwDF values (70%) was much higher than for LMwSDF (32%), whereas the variation coefficients were close for HMwDF and LMwSDF in wheat-based foods, 45% and 35%, respectively.

In the literature most of TDF content were obtained by the AOAC 985-29 method that essentially correspond to the HMwDF fraction determined with the AOAC 2009-01 method [START_REF] Stephen | Dietary fibre in Europe: current state of knowledge on definitions, sources, recommendations, intakes and relationships to health[END_REF], although resistant starch was not fully measured by the AOAC 985-29 method. Resistant starch content was low in our wheat-based foods (Table 1) and was not measured in the milling fractions, as it is known to be very low in unprocessed cereal samples.

TDF values were linearly related to HMwDF: TDFAOAC 2009-01= 0.989 HMwDF + 3.26 and very highly correlated (r 2 = 0.997) (Figure 1). The value of the intercept is close to the average value of LMwSDF calculated from the complete set of samples (3.0%). The measurement of the LMwSDF fraction has been subjected to debate and an evolution of the AOAC 2009.01 method has been proposed to correct an overestimation of the LMwSDF fraction in starch-rich food [START_REF] Brunt | Improvement of the AOAC 2009.01 total dietary fibre method for bread and other high starch containing matrices[END_REF][START_REF] Mccleary | Determination of total dietary fibre and available carbohydrates: A rapid integrated procedure that simulates in vivo digestion[END_REF]. Using only the data from the wheat-based foods (Table 1; the richer in starch and with overall lower TDF content) the intercept value of the relationship (e.g average LMwSDF content) was 2.06 while this value went up to 4.05 when only data from millstreams were used (Table 2. Supplementary Figure S1). Slopes and correlation coefficients of the relationships were not changed compared with the complete data set. In millstream fractions, starch content varied a lot compared to wheat-based foods, but the higher value of the intercept is not originating from an overestimation of the LMwSDF fraction due to starch. It is more likely due to the presence of fructans that represents 2 to 4% of wheat grain. These components are more abundant in bran [START_REF] Haskå | Distribution and characterisation of fructan in wheat milling fractions[END_REF][START_REF] Verspreet | Purification of wheat grain fructans from wheat bran[END_REF] than in white flour. Knowing that most of the data found in the literature were obtained with the AOAC 985-29 method corresponding to the values of HMwDF in this study, the TDF contents were in good agreement with values reported previously for similar wheat products and millstream fractions [START_REF] Elleuch | Dietary fibre and fibre-rich by-products of food processing: Characterisation, technological functionality and commercial applications: A review[END_REF][START_REF] Gebruers | Variation in the Content of Dietary Fiber and Components Thereof in Wheats in the HEALTHGRAIN Diversity Screen[END_REF]. Nevertheless values determined for fine/coarse bran were in a high range.

Dietary fiber content vs Arabinoxylans content

DF are a complex mixture of chemically heterogeneous carbohydrate components. In wheat grains arabinoxylans (AX) are the main polymers of the cell walls and therefore the main component of DF in wheat grain. Total AX contents varied on a quite large range between 1% and 30%, with similar trends within wheat product and millstream fractions as reported above for their TDF contents. Total AX content of wheat grain, and flour were comparable to previous reports [START_REF] Barron | Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition[END_REF][START_REF] Gebruers | Variation in the Content of Dietary Fiber and Components Thereof in Wheats in the HEALTHGRAIN Diversity Screen[END_REF][START_REF] Henry | Pentosan and (1 → 3),(1 → 4)-β-Glucan concentrations in endosperm and wholegrain of wheat, barley, oats and rye[END_REF][START_REF] Saulnier | Variability in grain extract viscosity and watersoluble arabinoxylan content in wheat[END_REF] but again values for coarse/fine brans were in a high range compared to literature.

The relationship between total AX content in the samples and TDF (or HMwDF) content is shown on Figure 2. A strong linear relationship (r 2 > 0.97) was established with nearly the same slope close to 2 between AX and TDF (TDF=2.15AX + 2.61; r 2 =0.975) or AX and HMwDF (HMwDF=2.17AX -0.57; r 2 =0.970). Using only the data from wheat-based foods (Table 1) or milling fractions (Table 2) had very little impact on the values of the slope, intercept or correlation coefficient of these equations (See supplementary Figure S2). Clearly, AX content corresponds to the HMwDF part of TDF. It is remarkable to note that, despite differences in AX structure and cell wall architecture within the different tissues of wheat grain, the AX content (simply measured as the sum of arabinose and xylose in the sample) accurately reflects the TDF contents of very different wheat products. Similar relationship between AX content of wheat samples and their dietary fibre content was previously reported: TDFEnglyst= 1.75AX -0.71 (r 2 = 0.994; 51 wheat samples) [START_REF] Bell | A rapid method of dietary fibre estimation in wheat products[END_REF]. In Bell's equation TDF content was determined by Englyst's method, based on the determination of the non starch polysaccharides and lignin content in the sample [START_REF] Englyst | Determination of the non-starch polysaccharides in plant foods by gas-liquid chromatography of constituent sugars as alditol acetates[END_REF]. The use of a different method for evaluating TDF content likely explains the differences in slope between the equation obtained in this work and by Bell [START_REF] Bell | A rapid method of dietary fibre estimation in wheat products[END_REF].

Dietary fiber content and tissue distribution

In millstreams, tissue composition was assessed through the biochemical marker methodology [START_REF] Hemery | Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions[END_REF] (Table 2). Pericarp (outer and inner) is mainly constituted of secondary cell walls [START_REF] Chateigner-Boutin | Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development[END_REF]. The testa is also constituted of secondary cell walls surrounded by a thick cuticular layer, whereas the nucellar epidermis is made of thin cuticular layer, and a tiny layer of low substituted arabinoxylans [START_REF] Barron | Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition[END_REF]. According to the dietary fibre definition, roughly all of these tissues, recovered in the outer pericarp and the so-called "intermediate layer", could be considered as dietary fibres. In the case of the endosperm tissues (the aleurone layer and the starchy endosperm), the TDF content could be approximated by the amount of cell walls in these tissues. In the aleurone layer approximately 45% of the dry mass is recovered in cell walls [START_REF] Hemery | Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions[END_REF]. Using the overall AX content in starchy endosperm or flour (around 2%) [START_REF] Barron | Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition[END_REF][START_REF] Gebruers | Variation in the Content of Dietary Fiber and Components Thereof in Wheats in the HEALTHGRAIN Diversity Screen[END_REF] and the proportion of AX in starchy endosperm cell wall, the amount of cell wall in this tissue was assumed to be 3%. TDF (expressed according to the dry matter) was calculated from tissue composition considering the following equation assuming equal distribution of water within the whole grain tissues: TDF (% dm) = %OP + %IL + 0.45*%AL + 0.03*%SE The calculated TDF was linearly correlated (r 2 =0.87) to the measured TDF with the following relationship TDFcalculated=1.07 x TDFmeasured + 0.25 (Figure 3). Regardless of the type of wheat or the millstreams a unique relationship was obtained in the whole TDF range (4-60%).

For a similar TDF content, variation in the proportion of the different grain tissue between milling fractions may affect their degradation by microbial enzymes and therefore their fermentation pattern e.g. aleuronic layer is easily degraded whereas the most outer layers of the grain are resistant to microbial attack (e.g. microbiota).

AX solubilisation by xylanase in relation with DF content and tissue distribution

The susceptibility to enzyme degradation of the different tissues of the grain has potentially important health impact as isolated AX polymers and AXOS are known to favour the development of beneficial bacteria [START_REF] Broekaert | Prebiotic and Other Health-Related Effects of Cereal-Derived Arabinoxylans, Arabinoxylan-Oligosaccharides, and Xylooligosaccharides[END_REF][START_REF] Neyrinck | Prebiotic effects of wheat Arabinoxylan related to the increase in bifidobacteria, roseburia and bacteroides/prevotella in diet-induced obese mice[END_REF]. Enzymatic hydrolysis with a xylanase is therefore proposed as a functional evaluation of DF in wheat fractions.

The amount of AX released by xylanase (AXenz) is reported in Table 1 and 2 and relationship with AX content is displayed on Figure 4. The amount of AXenz ranged from 0.7% up to 10% and was poorly related to the total amount of AX (r 2 = 0.64; Figure 4B). Especially samples with a high AX content (AX>25%) exhibited clearly large differences in the amount of AXenz. Figure 4A shows that the proportion of AXenz is globaly negatively correlated with AX (TDF) content (r 2 =0.57) e.g. the extent of AX solubilisation is lower for brans (see table 2; Figure 4A) and the peeling fractions and the highest for pure starchy endosperm fraction (flour T55 or semolina S3; see table 2). As expected, a better linear relationship (r 2 =0.73) was observed between the proportion of starchy endosperm and aleurone layer in a fraction and the proportion of AX released by the enzyme (see Figure S3).

Conversely, fractions very rich in outer pericarp tissue are little degraded by enzymes (e.g. fraction F -, debranning fraction A of common wheat). Therefore, the amount of AX solubilized from different fractions by xylanase treatment may be similar although these fractions may have very different AX (TDF) contents (e.g. F -fraction vs. F + fraction). In other words, the fermentation ability of wheat fractions is not simply related to AX/DF content and fractions with a high AX/DF content that have a lower degree of solubilization by xylanase may have more limited prebiotic effects. Such an assay can help to better understand the nutritional effects of wheat DF, which is not only related to their quantity. The ability of a fraction to modulate the microbiota is probably related to its fermentability (assessed by degradation with xylanase), but other characteristics such as particle size can also affect the microbiota, as recently demonstrated on wheat bran in a mouse model [START_REF] Suriano | Particle size determines the anti-inflammatory effect of wheat bran in a model of fructose over-consumption: Implication of the gut microbiota[END_REF].

Conclusion

In conclusion AX content can be used to estimate TDF content in wheat milling fractions and wheatbased foods. Moreover, the amount of AX released by xylanase is proposed as a functional evaluation of DF. AX solubilized by xylanase (AXenz) are also potentially releasable by microbiota enzymes in the intestine and the AXenz assay therefore indicates the proportion of fermentable DF in wheat milling fraction or in wheat-based foods. This assay provides a simple characterization of an important functional attribute of DF and could help to better understand the nutritional effects of different wheat milling fractions. The equation developed in this work is only valid for wheat products and cannot be applied to other cereal products such as corn, rye, barley, oat or rice… However we anticipate that the AX content could probably be used for the estimation of DF content in rye products, provided that an appropriate equation is developed. Indeed, in rye as in wheat, AX is the main component of cell walls and DF. The high amount of mixed linked-glucan (MLG) in oat and barley and the heterogeneity of the MLG distribution in the different parts of the grain are not favourable to the development of equations based on AX content to estimate the DF content, in these cereals.

Figure Captions

  's method distinguished soluble DF from insoluble DF but did not include the low-molecular-weight non-digestible oligosaccharides and neglected part of the resistant starch. Integrated methods were therefore developed for the determination of TDF, including non-digestible oligosaccharides and subsequently resistant starch by adopting physiological conditions for the removal of starch. The AOAC method 2009-01 (McCleary et al., 2010) and its extension the AOAC method 2011.25 (McCleary et al., 2012) measures the fraction of high molecular weight dietary fibre (HMwDF) by enzymatic-gravimetric techniques, and low molecular weight soluble dietary fibre (LMwSDF) by high-performance liquid chromatography (HPLC). HMwDF are actually formed of insoluble dietary fibre (IDF) and high molecular weight soluble dietary fibre (HMwSDF); HMwDF = IDF +

Figure 1 :

 1 Figure 1: TDF related to HMwDF contents in the wheat products (Data table1 and 2)

Figure 4 :

 4 Figure 4: A) AX amount released by xylanase (AXenz) and B) proportion of AX released by xylanase (% AXsol) related to AX content in the wheat products (Data table1 and 2)

Table 1 :

 1 Resistant starch and dietary fibre contents of wheat food products as determined byAOAC methods 2002.02 and 2009.01, respectively.

	g/100g d.m. Resistant Starch HMwDF LMwSDF TDF AXtot AXenz % AXsol
	Biscuit-R	0.14	1.8	1.3	3.2 1.0	0.7	67.4
	Biscuit-F +	0.17	7.2	1.8	9.0 3.5	1.4	40.1
	Biscuit-F -	0.17	6.4	1.3	7.7 3.2	0.9	27.3
	Bread-R	1.21	4.3	2.4	6.7 2.1	1.7	83.4
	Bread-F +	0.55	9.9	2.0	11.9 4.7	2.6	55.7
	Bread-F -	0.80	11.8	2.2	14.0 5.6	2.0	35.5
	Dry pasta-R	0.29	4.1	3.6	7.7 2.2	1.6	73.6
	Dry pasta-F +	0.21	8.0	2.9	10.9 4.1	2.2	54.4
	Dry pasta-F -	0.27	8.5	3.2	11.7 4.2	1.9	44.2
	Mean	0.42	6.9	2.3	9.2 3.4	1.7	53.5
	SD	0.37	3.10	0.80	3.3 1.4	0.6	18.6
	RSD	87%	45%	35%	36% 42% 37%	35%
	d.m.: dry matter
	R: regular bread, pasta and biscuits
	F + and F

-: products made with flour enriched in DF with F + and F -fractions SD : standart deviation ; RSD : relative standart deviation AXtot= sum of arabinose and xylose measured in the product AXenz= sum of arabinose and xylose released form the product after treatment with a xylanase % AXsol : = (AXenz/AXtot) x 100

Table 1 :

 1 Dietary fibre content, AX content and tissue proportion of various milling fractions from

	bread and durum wheats
	g/100g dm	HMwDF LMwSDF TDF AXtot AXenz % AXsol % OP % IL % AL % SE % EA
	Grain common wheat	14.9	3.4	18.3	8.1	3.1	38.5	2.5	3.7	9.2	79.9	1.3
	Coarse bran	52.4	4.6	57.0 25.0	9.9	39.6	13.3	29.4	36.0	15.5	0.5
	Fine bran	47.0	4.6	51.5 25.6	8.8	34.5	11.4	27.0	29.6	18.2	2.8
	Shorts	38.6	5.0	43.6 20.2	6.7	33.1	9.8	18.1	21.8	19.4	16.0
	Middlings	12.2	4.4	16.5	6.2	2.3	37.3	2.0	2.7	14.3	61.7	15.4
	Tail-end flour	7.6	3.8	11.4	4.0	1.6	39.3	1.2	1.3	11.5	76.7	8.7
	Flour (T55)	3.7	2.0	5.7	2.2	1.5	67.4	0.2	0.1	2.0	100.7	0.2
	Fibre fraction F +	20.1	3.9	24.1 10.0	4.7	47.1	3.9	9.1	20.9	56.7	3.8
	Fibre fraction F -	57.9	1.9	59.9 25.0	4.8	19.3	26.5	12.8	18.2	16.7	3.6
	Debranning fraction A	64.5	1.3	65.9 30.1	5.0	16.5	32.9	14.2	23.3	17.6	nd
	Debranning fraction B	46.1	2.5	48.7 24.5	7.7	31.4	15.2	15.8	27.4	27.3	nd
	Debranning fraction C	18.5	2.9	21.3 11.0	4.9	44.4	7.4	11.3	24.1	51.2	nd
	Debranning fraction D	25.6	3.0	28.6	8.3	4.1	49.2	4.9	8.1	20.4	63.1	nd
	Grain durum wheat	10.6	4.0	14.6	5.3	2.5	46.7	7.0		10.1	82.0	1.3
	Coarse bran	53.9	2.0	55.9 24.1	7.5	31.0	54.2		31.0	15.6	5.1
	Purified Fine bran	44.0	3.2	47.1 21.5	7.1	33.1	60.2		25.8	19.2	3.2
	Sized fine bran	49.7	2.8	52.6 25.4	5.2	20.4	51.7		27.4	18.2	2.0
	Shorts	18.0	4.1	22.2	8.7	4.7	53.7	16.9		21.8	56.1	5.1
	Tail-end flour	7.1	4.0	11.1	3.7	2.2	58.4	4.7		18.9	73.4	3.1
	Last break flour	6.7	3.8	10.5	3.4	1.8	52.1	4.0		18.3	78.7	0.8
	Semolina S6	7.4	4.2	11.7	3.8	2.3	59.6	0.8		12.0	81.9	6.6
	Semolina S3	3.1	3.6	6.7	1.8	1.5	83.0	3.6		2.4	100.0	0.0
	Debranning fraction E	69.5	0.9	70.4 28.6	2.3	8.1	67.5		12.5	10.8	nd
	Debranning fraction F	52.1	2.2	54.2 20.2	4.3	21.5	48.2		36.8	19.1	nd
	Debranning fraction G	45.5	2.6	48.0 19.4	4.6	23.5	55.4		33.7	25.6	nd
	Debranning fraction H	24.9	3.6	28.4 10.4	3.4	32.4	18.7		25.3	40.5	nd
	Debranning fraction I	15.5	3.6	18.9	8.6	3.0	35.1	11.1		17.8	62.5	nd
	mean	30.3	3.3	33.5 14.3	4.3	39.1	10.1	11.8	20.5	47.7	4.4
	SD	21.1	1.1	20.6	9.5	2.3	16.6	10.0	9.2	9.3	29.5	4.7
	RSD	70%	32%	61% 67%	54%	43%	99%	78%	45%	62%	106%
	d.m.: dry matter
	SD : standart deviation ; RSD : relative standart deviation
	AXtot= sum of arabinose and xylose measured in the product
	AXenz= sum of arabinose and xylose released form the product after treatment with a xylanase
	% AXsol : = (AXenz/AXtot) x 100
	OP: outer pericarp; IL: intermediate layers; AL: aleurone layer; SE: starchy endosperm; EA:
	embryonic axis

Acknowledgments

This work was funded by ANR: ANR-ALIA 2010 WHEAFI project.

The authors thank S. Daniel (BIA Nantes) for technical assistance in measuring AX content and performing xylanase degradation on the various wheat products. They also thank K. Mouacha, A.

Putois and T-M Lasserre (IATE Montpellier) for tissue composition analysis and G. Maraval (IATE Montpellier) for durum wheat mill streams production.

Supplementary figures

Supplementary figure S1: TDF content related to HMwDF content A) in the wheat-based foods (bread. pasta and biscuits; data