Surender Kumar Dhayal

Martin Lund

J M Van Den Brink

Ghouti Medjahdi
email: bghouti.medjahdi@univ-lorraine.fr

Alain Celzard
email: calain.celzard@univ-lorraine.fr

Vanessa Fierro
email: vanessa.fierro@univ-lorraine.fr

Carole Gardiennet
email: dcarole.gardiennet@univ-lorraine.fr

Andreea Pasc
email: eandreea.pasc@univ-lorraine.fr

Nadia Canilho
email: enadia.canilho@univ-lorraine.fr

Surender Kumar Dhayal

Enhancing the activity of biocatalysts supported on calcium phosphate by inducing mesoporosity with phosphopeptides

Keywords: biomaterial, biomineralization, hydroxyapatite-like nanoparticles, enzymatic supported catalyst

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Introduction

The wet precipitation of calcium and phosphate ions can lead to various calcium phosphate (CaP) polymorphs, including hydroxyapatite (Ca5(OH)(PO4)3H2O) [1]. The precipitation process starts with the nucleation and growth of primary particles, which can then aggregate into larger ones. Depending on the colloidal stability of the particles, inter-grains porosity can be induced, and in general, the higher is the stability, the higher is the porosity.

Still, such particles exhibit only a low surface area and low pore volume, especially at the mesoscale, which limits the use of calcium phosphate support as a enzymatic biocatalyst.

Strategies to tune the mesoporous texture in these inorganic particle matrices are based on controlling the crystal size and/or reducing the degree of crystallinity, either by using templates (e.g. based on self-assembled mesophases) to guide the crystal growth or by using polymeric coatings [2]. Much work on the immobilization of proteins on biocompatible supports such as porous silica for the development of enzymatic catalysts has also been published [3,4,5]. However, increasing the porosity of an inorganic support by coating it with organic food-grade polymers, for example, remains a straightforward approach compared to post-synthesis adsorption of the enzyme on a porous inorganic material. The coating strategy is much more suitable for bio-applications, especially for the elaboration of supported enzymatic catalysts. Indeed, both trapping and coating can be done in situ during the precipitation process of the inorganic matrix. Moreover, it is known that nucleation, growth and crystallinity of inorganic particles can be altered by the presence of a polymer in the synthesis medium [6,7]. The calcium phosphate nanoclusters (CaP) present in casein micelles are one example of particles stabilized by the organic phosphate groups of casein molecules. In general, casein adsorption is driven by interactions between the organic phosphate (serine-bound) and the surface of calcium phosphate particles. This adsorption leads to a 'brush-like' layer of casein polymers that improves colloidal stability and prevents the aggregation of nanoclusters. Furthermore, it has reported that the crystalline degree of calcium phosphate particles can be attenuated by the presence of biomolecule containing organic phosphate during the precipitation reaction [8]. Moreover, when proteins such as enzymes will be involved together with organic phosphate in the precipitation reaction, its will be trapped in the pores of the inorganic matrix [9]. The size of most food-grade enzymes is in the range 2 -20 nm, so the pore size of the inorganic matrix must be similar or slightly larger. Therefore, for the effective immobilization and retention of active molecules, both surface area and pore size must be controlled. In this study, we report the synthesis of supported biocatalysts by a one-step procedure involving wet precipitation and enzyme entrapment. An oligomeric or polymeric form of a phosphopeptide (e.g. casein glycomacropeptide or CGMP), extracted from whey, was added as a source of organic phosphate, to behave as a mesoporogen by modulating the crystallinity of the inorganic calcium phosphate matrix. The resulting materials were characterized in terms of porous texture, morphology and crystallinity. Additionally, DLS and 31 P NMR analysis were performed to better understand the formation mechanism of the hybrid inorganic-organic calcium phosphate particles. Finally, the catalytic efficiency of the biocatalyst support was determined as a function of the oligomer vs polymer nature of the glycopeptide.

Material and Methods

Materials

The casein glycomacropeptide (CGMP) was purified from a commercial sample (Lacprodan CGMP-10, Arla Food Ingredients, Denmark) [10][11][12][13]. After purification, the dry matter contained around 85 % protein of which about 73 % was monomeric CGMP (Mono-CGMP).

Polymeric CGMP (Poly-CGMP) was then prepared from this monomer by enzymatic crosslinking with microbial transglutaminase (mTG), following a procedure previously reported (see SI1) [14][15][16][17][18]. The enzyme (Enz), a lactase (Trade name Ha-Lactase, which activity is of 5200 NLU•g -1 of solution, namely 100k NLU•g -1 of protein), was provided by Chr.

Hansen A/S, Denmark. The lactase activity was measured by using o-nitrophenyl-bgalactopyranoside (ONPG) as substrate and the catalytic test was done according to the method of Kim et al. [19]. The following chemicals were purchased either from Sigma Aldrich for ONPG, dithiothreitol (DTT), calcium chloride dihydrate (CaCl2•2H2O) and magnesium sulphate heptahydrate (MgSO4•7H2O)) or from Merck for disodium hydrogen phosphate dihydrate (Na2HPO4•2H2O), sodium dihydrogen phosphate monohydrate (NaH2PO4•H2O), sodium chloride (NaCl), sodium carbonate (Na2CO3) and ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA). All were used as received.

The microbial transglutaminase (mTG) used for the enzymatic crosslinking of the monomeric CGMP was from Ajinomoto and had an activity of 1000 U per g of powder, as measured by the hydroxamate colorimetric method proposed by Folk et al. [20]. All other chemicals used and mentioned in this work were of analytical grade.

The morphology and microstructure of the powdered microparticles were analysed using Scanning Electron Microscopy (SEM, Hitachi microscope, Hitachi S4800). X-ray powder diffraction analysis was performed at 45 kV and 40 mA, with a wavelength of λCuKα= 1.5406 Å. The crystalline phases were identified by comparing the X-ray diffraction patterns with the JCPDS powder diffraction files produced by the International Centre for Diffraction Data.

The degree of crystallinity (DOC) of the samples was calculated from the ratio between the total crystalline area divided by the sum of total crystalline area plus total amorphous area extracted from the diffusion background. Then, the amorphous content (%) was determined from 1-DOC (see SI6c). The molar ratio of calcium (Ca) to phosphate (P) was determined by ICP-OES elemental analysis. to prepare CaP-Enz-Poly. In each case the final pH was 7. Those solutions were also added over a period of 5 min under stirring at 1000 rpm. After additional 5 min of stirring, the mixtures were allowed to rest for 30 min and then were stirred at 500 rpm for 2 h at 20 °C.

Synthetic route: wet precipitation

Mixing was stopped to allow particle sedimentation and slow growth and was continued gently after to avoid their agglomeration. At the end of step-(iii), the particle suspensions were centrifuged at 3000 × g (20 °C, 30 min). The pellets obtained after centrifugation were washed 3 times with 2L of MQ-water and dried at 20 °C using laminar airflow. The dried particles were hand grounded using a mortar and pestle and then stored in closed containers at room temperature. At the end of the step-(iii) 25 ± 3 g of dry powder was obtained from 1 L of starting suspension.

Loading and Activity of immobilized lactase

The amount of immobilized lactase in each sample was determined from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and bicinchoninic acid (BCA) calibration curves (see SI2). The enzyme loading (mg•g -1 ) was calculated from the ratio of the measured amount of protein (mg) to the weight of dry powder (g). The average mg•g -1 of enzyme loading and standard deviation were obtained from three replicates. Briefly, the powdered catalyst was suspended in 1 mL of PEM buffer (the same as Britton-Robinson buffer, see SI3 for details) at 10 g•L -1 . After separate incubation of catalyst and ONPG substrate solutions at 40 °C, the two preparations were mixed and stirred at 1000 rpm for 10 minutes at the same temperature of 40 °C. The reaction was then stopped by adding Na2CO3 (1M), followed by centrifugation at 10,000 × g for 5 minutes. The absorbance of the supernatant was measured at 420 nm. A blank measurement was done without any particles in the solution (see SI3). The measured activity was divided by the weight of the dry powder to obtain the specific activity of the material, in NLU•g -1 . The average NLU•g -1 and standard deviation were obtained from three replicates.

Textural characterization by nitrogen sorption

The BET area (ABET) and the pore size distribution (PSD) were determined using nitrogen adsorption at -196°C in an automatic equipment (ASAP 2020, Micromeritics) [21][22][23][24]. All samples were pre-treated before nitrogen adsorption by degassing under secondary vacuum at 25 °C for at least 48h. The parameters of the analyses are detailed in SI4.

Dynamic Light Scattering (DLS) on colloidal CaP in the presence of Mono-CGMP or

Poly-CGMP

The hydrodynamic radius (Rh) of the prepared colloidal CaP suspensions was measured at 25 °C using a Zetasizer (Nano ZSP, Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser. The intensity fluctuations in the scattered light were measured at 173° for 10 to 30 s. At least ten scans were averaged to obtain the correlation curve. The size distributions based on intensity diffusion were obtained using the general-purpose model in the standard Malvern software. To study the colloidal stabilization of CaP particles formed in the presence of Mono-CGMP or Poly-CGMP, two types of experiments were carried out without enzyme. In the first series, the concentration of the Poly-CGMP stock solution was fixed at 10 g•L -1 in a 50 mM Na-PB solution and the CaP particles were formed by one-pot precipitation by adding appropriate amounts of CaCl2 to reach a final concentration of 10, 50, or 100 mM, respectively. Then, the solution was rapidly transferred into the DLS instrument for analysis. The delay between the addition of CaCl2 and the start of analysis was one minute. In the second series of experiments, only Poly-CGMP solutions were prepared between 1 and 20 g•L -1 in a 100 mM Na-PB solution. The CaP particles were precipitated by adding a fixed amount of 2 µL CaCl2 (5M), e.g. 10 mM Ca 2+ as final concentration, to 1 mL of Poly-CGMP in Na-PB. The solution was kept at rest for 30 minutes at 25 °C, before the DLS measurements. The average and standard deviation were calculated from the data obtained from three replicates.

1 H and 31 P solid-state NMR

The experiments were performed on a Bruker Biospin Avance III spectrometer, at a magnetic field of 7 T corresponding to a 1 H resonance frequency of 300 MHz, using a 4 mm probe. The spinning frequency was set to 12.5 kHz, unless otherwise stated. A 300 s recycle delay was used for 31 P direct excitation spectra (single-pulse experiments). Proton to phosphorus cross-polarization (CP) 1D spectra were recorded with 1 H and 31 P resonance frequency fields of 47 and 34 kHz respectively, using a contact time of 1 ms. In twodimensional 1 H-31 P HETCOR correlations, proton to phosphorus-31 polarization transfer was achieved by CP with a 2 ms contact time. SPINAL-64 proton decoupling at 60 kHz was applied during 40 ms 31 P acquisition. 31 P and 1 H chemical shifts are relative to external 85% H3PO4 and TMS standards. Deconvolution and low spinning frequency spectra simulations were performed with dmfit software [25,26].

Results and discussion

CaP-based Biocatalyst Morphology

The main goal of this study was to investigate the formation of calcium phosphate-supported biocatalysts for food applications. For this purpose, organic phosphate sources (CGMP), namely phosphopeptides, were used to produce hybrid materials previously labelled CaP, CaP-Enz, CaP-Enz-Mono and CaP-Enz-Poly. Figures 1 andSI5 show the morphologies of the aforementioned systems (CaP, CaP-Enz, CaP-Enz-Mono and CaP-Enz-Poly), as obtained by scanning electron microscopy. Both pure CaP and CaP-Enz particles had platelets of brushitelike shape, which suggests that the enzyme has no influence on the formation of CaP particles (Figure 1a, 1b and Figure SI5). On the contrary, the addition of phosphopeptides significantly influenced the morphology of the resulting hybrid particles. Indeed, the platelets size decreased when using monomeric CGMP (Figure 1c) to completely disappear when using polymeric CGMP (Figure 1d). Moreover, aggregated, almost spherical nanoparticles appeared in the samples CaP-Enz-Mono and CaP-Enz-Poly. The sphericity of the particles seems to be better defined in the presence of the polymer compound and the size of the primary particles is around 50 -100 nm, which is close to the Rh of the pure polyglycopeptide in aqueous solution [27].

CaP particles formation followed by DLS

To decipher the formation of CaP particles, dynamic light scattering (DLS) analysis was performed in the presence of Mono-CGMP and Poly-CGMP to evidence their influence in the colloidal stabilization of CaP particles. As the synthesis is performed at pH 7 in the presence of Na2HPO4 as reagent, the precipitation of brushite (CaHPO4•2H2O) should be the most favourable solubility product constant (Ksp, of 2.6x10 -7 M 2 ). In the conditions chosen for DLS experiments, the ion activity product ([Ca 2+ ]x[HPO4 -]) is high and therefore the supersaturation might explain the rapid precipitation of CaP at room temperature (20 °C).

According to the DLS data (not shown), the pure CaP particles rapidly grew (within minutes) to a size larger than 1 μm and subsequently settled down under the influence of gravity. The same behaviour was observed for the CaP-Enz matrix, indicating that lactase does not act as a stabilizer either. In that case, the electrostatic repulsions from the surface charge are not high enough to prevent particle growth to large dimensions. In the case of Mono-CGMP and Poly-CGMP, different kinetics were observed. Figure 2a In the presence of Mono-CGMP (data not shown), the formation rate of CaP particles is slightly slowed down compared to pure CaP or CaP-Enz particles, which formed instantaneously. The monomeric CGMP does not seem to be an efficient particle growth inhibitor or colloidal stabilizer because large CaP particles (> 500 and 1000 nm) were formed within around 10 minutes in the presence of 10, 50 and 100 mM of Ca 2+ . Then, at longer time scales (> 10 min), a settlement phenomenon appeared. However, under the same conditions, CaP particles formation was found to be significantly delayed in the presence of the polyglycopeptide (Figure 2a). In fact, looking at the Rh values extracted from scattering intensity over time, below 10 minutes, the radius of the scattering objects has a size between 60 and 75 nm (corresponding to the P1 plateau), which corresponds to the Rh of the polyglycopeptide [27]. Then, suddenly, a new plateau of size around 90 -110 nm was reached (referred to as the P2 plateau) for any concentration of Ca 2+ . According to the representations of the intensity size distribution data (Figure 2b, 2c), the CaP clusters are stabilized by the polymeric CGMP, leading to highly monodisperse nanoparticles. This indicates that the Poly-CGMP provides much greater colloidal stability to the CaP particles than its monomeric form. Moreover, the size distribution representation for the P1 plateau overlaps the size range of the P2 plateau distribution. This could be related to ripening, which is a continuous process along the reaction time. Thus, the particle size distribution measured at a given time must comprise particles from both the initial stage and the ripened stage. These results obtained on the CaP and Poly-CGMP system seem to be similar to those observed in the case of CaP particles stabilized by Fetuin-A. Indeed, Fetuin-A is known to be an inhibitor of CaP mineralization and deposition in the soft tissues of animals and humans [28].

Wald et al. [28] found that in the presence of Fetuin-A, the CaP/Fetuin-A particles formed had initially a Rh of 40 -60 nm which ripened to a Rh of around 100 nm after a lag period of about 300 minutes. They also argued that particle size was mainly governed by the concentrations of the mineral ion and Fetuin-A. Increasing the concentration of Fetuin-A led to smaller particles, while increasing the concentration of the mineral ion accelerated the particle ripening process at fixed amount of Fetuin-A. Thus, they proposed that the size and stability of the 'calciprotein' particles could be controlled by a combination of these physicochemical parameters. It should be noted that Fetuin-A has several phosphoserine residues and O-and N-glycosylated chemical groups (see e.g. P02765 in www.uniprot.org).

In our case, the monomeric CGMP prepared from k-casein phosphorylated serine residues and the polyglycopeptide synthesized from the monomeric form is a polyelectrolyte. Therefore, it has a higher density of phosphoserines per molecule, which allows binding to the surface of inorganic CaP in a way analogous to caseins micelles for CaP nanoclusters [29]. Moreover, by analogy with Fetuin-A, the polymeric CGMP also has O-glycosylated residues, which are known to control the size and stability of CaP particles by varying the Poly-CGMP concentration, as presented in Figure 3. Thus, according to Figure 3, the RH values (measured after 10 min of reaction) decrease with increasing Poly-CGMP amount in the solution. Then, above 10 g•L -1 , the CaP particle size is stabilized at a hydrodynamic radius of around 100 nm. This clearly demonstrates that the polymeric CGMP acts as a colloidal stabilizer, and it can be speculated that the mode of action is a combination of electrostatic and steric repulsions originating from a combination of a charged backbone and a higher molar mass of the polyelectrolyte. The threshold of 10 g•L -1 can be designated as the maximum surface concentration of the polymer adsorbed on the CaP particles formed.

Textural and structural properties

Nitrogen adsorption isotherms have led to a better understanding of the textural properties of the dried materials (see SI4 Table andFigures). The results show that the specific area (ABET) and the mesoporous volume (Vmes) increased in the case of CaP-Enz-Mono and CaP-Enz-Poly particles compared to CaP and CaP-Enz: 70 and 59 m 2 •g -1 vs 36 and 31 m 2 •g -1 for ABET, respectively, and 0.218 vs 0.150 cm 3 •g -1 in average, for Vmes, respectively. This could be due to voids formed between aggregated nanoparticles or, in other words, due to interparticle porosity. In the case of platelet-shaped particles, these voids are almost negligible at the mesoscale. On the contrary, less ordered aggregated nanoparticles, trapped in an organic network, might induce the observed mesoporosity (Table SI4). Furthermore, the pore size distribution (PSD) became much more monodisperse in the presence of Mono-CGMP or Poly-CGMP, and it was centred on 15 nm (Figure SI4 a,b). These results highlight the fact that the porous texture in the mesopore range (2-50 nm) of the inorganic carrier matrix can be tailored by the monomer or the polymer to obtain pore sizes suitable for enzyme entrapment (herein Ha-Lactase, 14 nm in size). In order to understand whether the CGMP (monomer or polymer) interferes with the inorganic particles in the crystallisation process, the variation of crystallinity in the CaP, CaP-Enz, CaP-Enz-Mono and CaP-Enz-Poly samples was measured by XRD (See 4a,b). Thus, the platelet-shaped particles observed for the CaP or CaP-Enz systems were clearly identified as monoclinic, prismatic crystals of brushite (CaHPO4•2H2O). This result agrees with the existing literature. For instance, under neutral or slightly acidic conditions, wet precipitation of CaP particles leads to the rapid formation of a platelet-like brushite phase (see Fig. 4a) [2]. The intensity and resolution of the crystal peaks corresponding to brushite significantly decreased in the case of CaP-Enz-Poly. The broadened pattern obtained for CaP-Enz-Poly is a typical signature of a more disordered calcium phosphate phase such as reported by Sun et al. [30].

However, the most pronounced crystal peaks still present in the signal of the CaP-Enz-Poly sample mainly fitted the hydroxyapatite (Hap) crystalline form (Ca10-x(PO4)6-x(HPO4)x(OH)2-x) (see Fig4b). Finally, the ratio between the areas under the broadened bands and the sharp peaks gave an estimate of the degree of disorder in each sample prepared. Thus, it appears that the proportion of disordered phase in the CaP particles increased from 15% without CGMP to around 45 % in the presence of the phosphopeptide [7]. This clearly indicates that polymeric CGMP disturbed the crystallization process of CaP nanoparticles. Indications on the nature of phases can also be obtained from the Ca/P molar ratio determined by elemental analysis (see Table SI7). Indeed, it is known from the literature that the Ca/P molar ratio can vary between 1.5 and 1.67 for Hap and calcium-depleted Hap, while it is 1 for brushite and varies from 1.2 to 2.2 for amorphous calcium phosphate, respectively [1,2]. A Ca/P molar ratio of 1.08 was determined in the case of CaP particles, thus confirming the presence of brushite, and of 1.27 in the case of CaP-Enz-Poly particles (1.25 for CaP-Mono), which could correspond to amorphous Hap (see SI7).

Solid-state NMR

Solid-state NMR (ssNMR) was used to further characterize the different types of phosphates and phosphorus environments in CaP, Cap-Enz, CaP-Enz-Mono and CaP-Enz-Poly. Direct 31 P excitation with a long recycle delay allows quantitative analysis of different phosphoruscontaining species, whereas cross-polarization (CP) experiments enhance the 31 P signals of near-proton and immobilized phosphorus atoms, as shown in Figure 5. 31 P chemical shifts as well as the relative signal intensity in the two types of 1D spectra enabled a first identification of different phosphorus species.

Additional information was obtained by recording the 31 P low spinning frequency spectra and simulating these spectra with different anisotropy parameters. The experimental details and simulations are given in SI8. The 1D 31 P spectra of CaP, CaP-Enz, CaP-Enz-Mono and CaP-Enz-Poly samples (Figure 5) exhibit two major peaks at 3-3.3 ppm and 1.5 ppm. An additional signal around -0.2 ppm is also present, excepted for CaP.

The component at 1.5 ppm corresponds to protonated phosphates according to chemical shifts reported in the literature. Its protonation state is confirmed by the high relative intensity in the CP spectra. Its contribution is similar in CaP and CaP-Enz, 56% and 51%, respectively, but largely decreases in the presence of mono-or poly-CGMP (see Tables SI8-1, SI8-2, Fig. SI8-1 and Fig. SI8-2). CaP, CaP-Enz, CaP-Mono spinning sidebands manifolds in 1D 31 P low spinning frequency CP spectra, can be simulated using daniso and h values of 62 ppm and 0.8, respectively (see Fig. SI8-3 and Tab. SI8-3). These parameters are typical for brushite [31][32][33]. The broader signal of CaP-Enz-Poly is more difficult to deconvolute but gives an daniso value around 45 ppm and is therefore probably not related to brushite (SI8-4).

These observations are consistent with the XRD data. Confirmation of brushite phase comes from 2D 1 H-31 P correlations: strong cross-signals between 31 P at 1.5 ppm and 1 H at 10.7 ppm in CaP-Enz are indeed typical for brushite (see SI8.5) [34,35]. The 31 P signal at 3-3.3 ppm is broad (2 to 2.5 ppm FWHM) and its relative intensity is lower in CP spectra. The contribution of this peak to the total 31 P signal is 44% in CaP and CaP-Enz samples, and 65% in CaP-Enz-Mono and 67% in CaP-Enz-Poly samples (see Table SI8.1). Its anisotropy contribution is not visible in the low spinning frequency experiments of CaP and CaP-Enz, whereas in CaP-Mono and CaP-Enz-Mono it shows an anisotropy parameter around -20 or -30 ppm (see Figure SI8.4), consistent with inorganic colloidal calcium phosphate in a hydroxyapatite-like environment. In the 2D 1 H-31 P spectra of CaP-Enz-Poly, a correlation between the 31 P signal at 3-3.3 ppm and a 1 H hydroxyl group resonance at -0.2 ppm is again a hallmark of hydroxyapatite [26,31,36]. This correlation is more intense in CaP-Enz-Poly, in agreement with XRD data indicating a hydroxyapatite phase representing 96% of the crystalline phase (data from area integration of crystal peaks, not shown). The C-terminal peptide of k-casein is flexible and is known to remain a flexible tail in casein micelles, in contrast to aand b-caseins, which participate in micelle formation and show an immobilized phosphoserine (SerP) 31 P signal [35]. Therefore, monomeric CGMP should not interact extensively with calcium phosphate particles. The 31 P data of CaP-Mono and CaP-Enz-Mono (brushite signal, only a small amount of Hap-like particles) are closer to CaP and CaP-Enz than to CaP-Enz-Poly. On the contrary, the CaP-Enz-Poly sample exhibits a higher proportion of Hap-like phosphate. The broad 31 P signal of CaP-Enz-Poly around 1.8-2 ppm present in 1D CP spectra (left peak, Figure 5) and in 2D HETCOR (SI8-5) correlation can be tentatively assigned to immobilized phosphoserine from poly-CGMP [35]. The immobilization of these SerP groups could be ascribed to their participation in the interaction with calcium phosphate particles [35]. Peptide immobilization, and thus SerP participation in colloid formation, seems only observable in the presence of polymeric CGMP.

The detailed characterization of the inorganic particle seems to indicate that the presence of peptide leads to less crystalline particles (XRD) and to the appearance of hydroxyapatitelike calcium phosphates (XRD and NMR). The immobilization of the peptide phosphoserine groups by participation in the interaction with the calcium phosphate particles has been tentatively evidenced by NMR only for CaP-Enz-Poly. The phosphoglycopeptide polymer appears to be necessary to allow enzyme retention with good activity, to increase the proportion of hydroxyapatite-like particle and to prevent the formation of the brushite phase.

Enzyme activity of the Hap-like catalytic supports

The Figure 6 compares for the different samples: i-the amount of lactase immobilized in the different samples prepared by in situ wet precipitation as described above, and ii-the respective catalytic activities. Thus, the CaP-Enz and CaP-Enz-Mono particles contained respectively 52 ± 19 and 34 ± 21 mg of immobilized lactase per g of dry powder, while the CaP-Enz-Poly particles had an amount of encapsulated lactase of 125 ± 4 mg•g -1 , which is more than twice the amount immobilized in the CaP-Enz and CaP-Enz-Mono matrices. The amount of enzyme immobilized in CaP-Enz and CaP-Enz-Mono particles is of the same order of magnitude as that typically observed for silica matrix (10 -70 mg•g -1 ) [9]. In addition, according to the standard deviation values, the lactase encapsulation process seems to be highly reproducible in the case of CaP-Enz-Poly particles compared to the other two samples.

Consequently, the supported enzyme presented variable catalytic performance, as also shown in Figure 6. It can be seen that the activity of the CaP-Enz-Mono sample is about three times higher than that of the CaP-Enz sample, despite the similar amounts of immobilized protein. However, the activity of the CaP-Enz-Poly sample was found to be almost 10 times higher than that of the CaP-Enz material. The average specific activity was found to be 1060, 4811 and 4843 NLU•g -1 of protein for CaP-Enz, CaP-Enz-Mono and CaP-Enz-Poly samples, respectively. The specific activity of the immobilized enzyme in the CaP-Enz sample was found to be around 100 times lower than that of the free enzyme (100 kNLU•g -1 of protein), while it was only 20 times lower in the case of CaP-Enz-Mono and CaP-Enz-Poly samples. A comparison of the specific activity of immobilized and free enzyme phenomenologically indicates the hindrance to activity due to slower mass transport of the substrate and product through the inorganic matrix. This suggests significant resistance to mass transfer (diffusion) through the particles. The narrowest PSD found for the CaP-Enz-Poly particles could explain the better retention and the higher activity of enzyme in CaP-Enz-Poly particles compared to the other two types of particles. The differences in enzymatic activity between the materials CaP-Enz, CaP-Enz-Mono and CaP-Enz-Poly might be due to some differences of particles mesoporosity as well as to enzyme denaturation caused by adsorption on the inorganic matrix surface. Indeed, a direct interaction of the enzyme with the surface of pure CaP might lead to a partial denaturation of the enzyme, e.g. due to some changes in the quaternary/tertiary structure. A specific activity about five times higher for CaP-Enz-Mono and CaP-Enz-Poly than for CaP-Enz particles indicates some protective effect in the case of monomeric and polymeric CGMP. However, CaP-Enz-Poly had a much higher total activity (607 ± 34 NLU.g -1 of dry powder) than monomeric CGMPcoated particles (161 ± 70 NLU.g -1 of dry powder), which indicates that the difference between the activities of these two types of particles is mainly caused by the different amounts of encapsulated enzyme (Figure 6). This suggests that the monomer or polymer preferentially adsorbs on the CaP surface and that the enzyme is therefore mainly trapped in the organic network where the quaternary/tertiary structure of the lactase might be better preserved. Similar observations were made on lactase immobilized in silica matrix, where the enzyme encapsulated in macropores was found to be more active than the enzyme adsorbed in mesopores [4]. The authors proposed that the difference in activity was related to the quaternary structure of lactase (monomers vs dimers or tetramers).

Conclusion

In conclusion, through the careful selection of precipitation conditions, phosphopolypeptide polymers have been found to be efficient controllers of crystal growth, crystallinity and colloidal stability of calcium phosphate particles (CaP) when produced by a wet precipitation method. Changes in the crystalline phase, from brushite to hydroxyapatite, crystal size and disorder led to changes in the mesoporous texture of these particles and made them suitable for trapping small bioactive macromolecules such as enzymes while preventing significant loss of catalytic activity. The polymeric form was found to be more suitable for encapsulation since it produces a good interaction with the CaP surface and imparts excellent colloidal stability to the particles. Based on this mechanism, it can be concluded that any bio-macromolecule with organic phosphates and flexible structure can be used to modulate the mesoporosity of calcium-based inorganic particles. The underlying fundamental mechanism seems to be the ionic interactions between the charged macromolecule and the charged inorganic surface. Thus, extending the proposal further, any (bio)macromolecule with strong ionic groups present in its chain that can ionically interact with an inorganic surface should modify its mesoporous texture. Of course, there could be variations originating from other parameters such as the flexibility of the chain, the distribution of charged groups and the presence of other groups that interact with the surface via other types of weak interactions. Using biopolymers for controlling/modulating the mesoporous texture of inorganic matrices seems to be a viable option for practical applications. Modified mesoporosity with a suitable pore size can be also used for the

Conflicts of interest

There are no conflicts to declare.

Four

  materials have been synthesized by wet precipitation. The sample CaP refers to the pure inorganic matrix of calcium phosphate (CaP) prepared without enzyme (Enz) and CaP-Enz with enzyme. The hybrid particles obtained in the presence of enzyme and mono-CGMP (Mono) or poly-CGMP (Poly) have been abbreviated as CaP-Enz-Mono and CaP-Enz-Poly, respectively. The following stock solutions were used for preparing the different samples mentioned above: (a) 0.1 M sodium phosphate buffer and 0.1 M NaCl at pH 7.0, named Na-PB, (b), 4.5M CaCl2, (c) 0.3M Na2HPO4, (d) 60 g•L -1 of Mono-CGMP in Na-PB, (e) 20 g•L -1 of Mono-CGMP in 0.3 M Na2HPO4, (f) 60 g•L -1 of Poly-CGMP in Na-PB, and (g) 20 g•L -1 of Poly-CGMP in 0.3 M Na2HPO4. The lactase enzyme stock solution (Enz) was used as received and MQ-water (W) was used in all stock solutions. The preparation of the different samples involved three sequential steps. Step-(i): Prepare 940 mL solution by mixing equal volumes of W and Na-PB (a) for CaP sample; Enz and Na-PB (a) for CaP-Enz sample; Enz and 60 g•L -1 of Mono-CGMP (d) for CaP-Enz-Mono sample; Enz and 60 g•L -1 of Poly-CGMP (f) for CaP-Enz-Poly sample. Step-(ii): 60 mL of CaCl2 stock solution (b) were added dropwise for 5 min under 1000 rmp to 940 mL solution obtained in step-(i). Step-(iii): Addition of the resulting mixtures to 1L of respectively 0.3 M Na2HPO4 in the case of CaP and CaP-Enz samples, 20 g•L -1 of Mono-CGMP stock solution (e) for the CaP-Enz-Mono sample; and 20 g•L -1 of Poly-CGMP stock solution (g)

  depicts the variation of the hydrodynamic radius (Rh) during the formation of CaP particles over time, at three different concentrations of Ca 2+ ions, i.e., 10, 50 and 100 mM, in the presence of Poly-CGMP at a concentration of 10 g•L -1 .

  immobilization of various bioactive molecules, macromolecules or cells targeting straightforward applications for food packaging, (ref https://doi.org/10.3390/polym13234198) pharmaceutics (ref notre Adv Colloids and Interface science) or functional food (ref https://doi.org/10.1111/1541-4337.13049).

Figure 1 .

 1 Figure 1. SEM images of dry particles showing platelets (a) CaP, (b) CaP-Enz, or nanoparticles

Figure 2 .

 2 Figure 2. Mean hydrodynamic radius (RH) of the CaP particles formed in the presence of Poly-

Figure 3 .

 3 Figure 3. Mean hydrodynamic radius (RH) of the CaP particles formed in the presence of

Figure 4 :

 4 Figure 4: XRD showing the presence of brushite in the CaP sample and the formation of highly

Figure 5 .

 5 Figure 5. 1D 31 P NMR spectra of CaP (black), CaP-Enz (blue), CaP-Enz-Mono (green) and CaP-

Figure 6 .

 6 Figure 6. Amount of immobilized lactase (mg•g -1 of material) and enzyme activity (NLU•g -1 of

  Figure 1

Figure 4 .

 4 Figure 4.

Acknowledgements

SKD et al would like to thank Mr. Jean Cauzid for SEM analysis and Mr. Jérôme Marin for elemental analysis measurements.

Funding

This work was supported by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA (grant agreement n° 606713). A.C and A.P. acknowledge financial support from the "Impact Biomolecules" project of the "Lorraine Université d'Excellence" (Investissements d'avenir -ANR 15-004).


Laurent Thiong-Kay

Nikos Smyrnaios

Radical Journalism à la française. Between Differen6a6on and S6gma

Focusing on the case of French radical journalists, this chapter has several objec7ves. A:er recalling the specific context of repoli7ciza7on of the journalis7c field in France, under the influence of digital media, it focuses on the condi7ons that allow for certain journalists to break with the professional ideology of neutrality-objec7vity. In a second step, our analysis specifies why this break is par7al. Indeed, our interviewees remain aEached to the journalis7c deontology as the basis of their professional legi7macy. They specify the func7on of journalism against the compe7ng figures of the 'automedia' and the 'editorialist' Finally, they are aware that commiEed and radical journalism is a label or a s7gma that can reinforce the mistrust toward them by the poli7cal and media elites.

Introduc6on

. At the same Jme, journalists became increasingly precarious and were subjected to intense compeJJon for material but also symbolic resources such as the 'press card'. The aZribuJon of this card is supposed to disJnguish professional journalists, who are meant to respect deontological norms such as rigor, accuracy, integrity, fairness, imparJality and objecJvity, from other media workers, outsiders, radicals or militants. Thus, the press card funcJons as a mechanism of symbolic recogniJon of the profession -and therefore exclusion of media perceived as non-mainstream.

It was not unJl the end of the 1990s and the anJ-globalizaJon movement that this trend was strongly and concretely challenged. These mobilizaJons sought to find margins of autonomy vis-à-vis mainstream media. Based on the Indymedia model, a wave of "mediacJvism" [START_REF] Cardon | MédiacJvistes[END_REF] developed in France. Symmetrically, the criJcism of mainstream media spread to professional journalism itself: numerous outlets that carry out an endogenous criJque of the media industry were created. The development of online news from the early 2000s onwards has allowed the rise of alternaJve and parJcipatory media that could reach a wider public with relaJvely low costs [START_REF] Marty | Diversité et concentraJon de l'informaJon sur le web: Une analyse a grande échelle des sites d'actualité français[END_REF][START_REF] Smyrnaios | La producJon médiaJque de l'opposiJon au barrage de Sivens sur Internet, entre reconfiguraJons info-communicaJonnelles et repoliJsaJon de l'enjeu local[END_REF]. Since that period, many original editorial projects have developed, engaging themselves in promoJng solidarity, ecology and progressive poliJcs [START_REF] Lévêque | Journalistes engagés, Presses universitaires de Rennes, séries[END_REF].

Our research aims to understand the sociopoliJcal logics behind the emergence of this alternaJve and someJmes-radical journalism. More precisely, we want to quesJon the idenJty of poliJcally commiZed journalists and their relaJonship to the norms of their profession. Our objecJve is to examine the condiJons that make the advent of this new professional idenJty possible. In order to do so, we composed a sample of six journalists who are freelance or have worked as permanent staff for five independent professional online media with a large audience: Grégoire Souchay, who works for Reporterre, a news website sympatheJc to ecologist and alternaJve movements founded by a former journalist of Le Monde; Camille Polloni, who worked for Basta!, an online media with a team of journalists and civil society acJvists and Les Jours, an independent subscripJon-based news website founded by former journalists of LibéraJon; Louise Fessard and Jade Lindgaard, who work for Mediapart, a progressive subscripJon-based invesJgaJve journalism website founded by a former director of Le Monde; Gaspard Glanz founder of the press agency Taranis News, which documents social conflict from the inside; and Frédéric Scheiber, a freelance photographer who has worked for several independent outlets covering parJcularly social movements.

Through lengthy semi-structured interviews with these journalists and content analysis, we studied their producJons, their trajectories and their representaJons in the context of a qualitaJve study on the media coverage of the movement against the Sivens dam (Thiong-Kay, 2020a,). This mobilizaJon is part of the struggles against what is called in France 'Grands Projets InuJles et Imposés', a designaJon that groups together massive construcJon projects that are considered anJ-ecological, inefficient and useless such as the Notre-Dame-des-Landes airport in the region of Nantes, the Center Parks in Roybon or the Bure radioacJve waste burial project.

The Sivens dam (Barrage de Sivens) was a dam which was planned for construcJon across the Tescou, a tributary of the Tarn River in southwest France, near Toulouse, that aimed to support intensive agriculture. Supported by local authoriJes such as the Department of Tarn, the dam was to be built in the Testet wetland which includes an extremely rich ecosystem, in the heart of a forest that is the "subject of aZachment" [START_REF] Latour | Où aZerrir? Comment s'orienter en poliJque[END_REF], in the region. In 2011, a collecJve called "Tant qu'il y aura des bouilles" was created to fight against this project. Later, hundreds of people came to protect the biodiversity of the wetlands and to prevent the construcJon of the dam, creaJng a permanent protest camp in 2013 the Testet ZAD (Zone à défendre or 'Zone to Defend'). The Testet ZAD was inspired by one of Notre-Dame-des-Landes, the most wellknown 'Zone to Defend' in France that resisted several concerted aZempts by the French state to evict it in order to construct a new airport.

The sociological composiJon, ideological background and repertoires of collecJve acJon [START_REF] Tilly | ContenJous repertoires in Great Britain[END_REF] of the two groups opposing the Sivens dam differed: the first one was composed mainly by ecologist militants and was legalisJc [START_REF] Ollitrault | Sous les pavés, la Terre. MobilisaJons environnementales en France[END_REF]; the second one included anarchists and radical ecology acJvists and favored direct and someJmes illegal acJon. ConstrucJon work began in 2014 and was then halted aper Rémi Fraisse, a 21-year-old man protesJng against the construcJon project, was killed by a stun grenade fired by police. His death sparked further protests across France, some of which were violent. The project was then abandoned in 2015. This movement was covered by the journalists who we interviewed for this research.

Informa6onal Ac6vism and Fragmenta6on of the Journalis6c Field

At the heart of the rise of radical journalism in France, we find journalists with a poliJcal trajectory and socializaJon that predispose them to forms of, if not commitment, at least criJcal reflexivity. Most of the respondents have a university educaJon in social sciences (sociology, poliJcal science) which gives them the ability to quesJon the characterisJcs of their profession and their own relaJonship to it. On the other hand, the creaJon of lep-wing media, in the context of the restructuring of the journalisJc field [START_REF] Demers | DéstructuraJon et restructuraJon du journalisme[END_REF], allows poliJcal radicalism to flourish in the media. Our respondents have an acJvist experience that gives a specific meaning to their journalisJc pracJce. But it is necessary to work for certain media so that this poliJcizaJon can be fully combined with journalism.

For example, Jade Lindgaard graduated from Sciences Po Paris and the Centre de FormaJon des Journalistes in 1997. She started working for AFP, the French public press agency, in the economics department, which she then lep for the cultural magazine Les InrockupJbles, where she worked on contemporary art unJl 2007. But Jade Lindgaard then experienced personal acJvism in the anJ-globalizaJon movement through the climate acJon camps. She recalls:

[…] A revela9on about the importance of the climate issue and the richness of the climate jus9ce movement. I see this thing, both the radicalness of the proposal, the crea9vity of the mode of mobiliza9on, the intellectual richness of the exchanges, how the very sharp exper9se on energy transi9on plans.

Jade Lindgaard, Mediapart

Back in France, she set out to cover the climate issue. This objecJve coincided with her recruitment at Mediapart, an online invesJgaJve media explicitly posiJoned on the lep of the poliJcal spectrum:

I was offered] a posi9on in higher educa9on and research, and I told him [Mediapart's editorial director] that I was really interested, but I asked him what he had planned for ecology […]. He looked at me and replied: "Nothing, for me ecology means turning off the tap water when we brush our teeth, that's fine.]" [It was] completely anecdotal in his eyes. I was very upset when he said that. And then I remember well, he looked at me and said: "If you also want to do ar9cles on ecology, of course it will be possible". And so, it started like that. To answer your ques9on, it was also, at the beginning, a fight within Mediapart to impose ecology as a subject in its own right. […] Today it would never occur to anyone in the editorial staff to ques9on the legi9macy of having a full-9me person on ecology.

Jade Lindgaard, Mediapart

The case of Gaspard Glanz (Taranis News) is also enlightening. Socialized in parJcipatory democracy within the Strasbourg youth council, he is strongly commiZed during his university years against the two neoliberal laws promoted by right-wing Presidents Jacques Chirac, in 2006, and Nicolas Sarkozy, in 2009: respecJvely, the "first employment contract" and the law on the "freedom and responsibiliJes of universiJes". There he rubbed shoulders with the fringes of autonomous university acJvism, and quickly experienced clashes with the police. At the heart of the confrontaJons, he became familiar with filming from a first-person point of view (Thiong-Kay, 2020b), which was part of a more general trend of internet acJvists [START_REF] Blondeau | Devenir Média. L'acJvisme sur Internet, entre défecJon et expérimentaJon[END_REF]. At that point, for Gaspard Glanz, the objecJve was less to publicize the conflict than to build up a corpus of images that can defend the acJvists in case of a trial [START_REF] Nez | Des informateurs citoyens. Usages des images par les indignés espagnols[END_REF]. He once again indulged in the pracJce at the NATO summit in Strasbourg in 2009, marked by the first appearance in France of the "black bloc", a tacJc used by protesters who wear black clothing and other face-concealing and face-protecJng items associated with anarchism. At the heart of the protest, the then acJvist understood that there were no journalists to film these demonstraJons from the inside. He then saw the potenJal of such a pracJce as a professional journalist:

And it's also the first 9me I'm in a place where there are no journalists. I'm in my city, I'm at the heart of the demonstra9on, of the "bloc" as it could exist at the 9me, without a banner, without anything, but I'm embedded in the middle of the demonstrators, and while demonstra9ng, I don't have a TV headset, I just have a camera. […] It was the real turning point, April 2009, when I was no longer an ac9vist in the sense of being ac9ve on the ground.

Gaspard Glanz, Taranis News

It is therefore in his acJvist commitments that Gaspard Glanz draws the specificity of his journalisJc acJvity. He finished his studies in sociology and created his company in Rennes in 2011. IniJally planned to be a subsidiary of Rue89's network of local news websites, the project was abandoned and then transformed to a TV agency and renamed Taranis News. The agency's journalist started working on the subjects of Notre-Dame-des-Landes and Sivens Zones to defend, the situaJon of migrants in Calais, and then the Yellow Vests movement, a series of grassroots weekly protests for economic jusJce and insJtuJonal poliJcal reforms that began in France in November 2018. Of course, the subversive and someJmes ambiguous nature of Taranis News' reports and publicaJons earned our interviewee hosJlity. This hosJlity comes not only from the poliJcal field, but also from his fellow journalists, as we will see later.

The French journalisJc field is indeed characterized by fragmentaJon, power struggles and legiJmacy disputes that are exacerbated by its depoliJcizaJon. Thus, commiZed journalists have to work in independent radical media in order to fight for the recogniJon of certain issues they care about because poliJcally controversial topics are open neutralized by the mainstream press. This is parJcularly true of climate change. As Jean-BapJste [START_REF] Comby | La quesJon climaJque. Genèse et dépoliJsaJon d'un problème public[END_REF] has shown, the dominant, generalist naJonal media do not offer regular forums to social actors criJcizing the ecological damage provoked by capitalism. This situaJon is considered structurally unfavorable by our respondents, which leads them to invest themselves in the margins of the journalisJc field. However, this "migraJon" is not an exit, but on the contrary a will to influence the profession and push the media to parJcipate in the emergence of public problems and their inclusion in the poliJcal agenda [START_REF] Hassenteufel | Les processus de mise sur agenda: sélecJon et construcJon des problèmes publics[END_REF]. For our interviewees, this requires the consolidaJon of their media ventures. It is therefore a quesJon of fighJng not only 'through' the media but also 'in' the media.

My ac9vism includes poli9cs, ecological and social commitment, but it is above all how to allow other media to exist in the journalis9c universe. So that we can get out of the niche. As François Ruffin (French journalist who is now deputy of the Le\) says: "alterna9ve media are not an alterna9ve".

Grégoire Souchay, Reporterre

CommiZed journalists thus assert their desire to treat informaJon differently. By seeking to change their 'framing' or 'angle', they have a twofold objecJve: to offer original coverage in order to establish the specificity of their media, but also to deploy a counter-framing to the representaJons of the social world in force within the dominant media space formed by the naJonal and regional press, as well as poliJcal communicaJon. During the latest social movements in France, stereotypes were aimed at the subaltern counter-publics [START_REF] Fraser | Repenser l'espace public: une contribuJon a la criJque de la démocraJe réellement existante[END_REF], such as the 'zadists', the 'black blocs' or the 'Yellow Vests':

[On the subject of the environmentalist mobiliza9on against the Sivens dam] We also had this concern at Rue 89 to shi\ the gaze a lible in rela9on to what could be done in the daily press […] We agreed on the fact that it was more interes9ng to tell where the people who were demonstra9ng there came from, what their intellectual influences were, their militant backgrounds, and what they came to find on the ZAD.

Camille Polloni, Les Jours

Finally, radical journalism also comes about as a reacJon to what the actors perceive as the dysfuncJons of the mainstream media. These dysfuncJons have prompted them to join the poliJcized margins of the field, which carry out a criJque in acJon of the laZer. Thus, the strong dependence of the regional daily press on local poliJcal actors [START_REF] Bousquet | Pour une approche globale de l'informaJon infranaJonale, HabilitaJon a Diriger des Recherches en Sciences de l'InformaJon et de la CommunicaJon[END_REF] and insJtuJonal sources is pointed out: These are journalists who will enter the police sta9on much more easily. They have a rela9onship of trust with the police officers, who are also their suppliers of … of news in fact.

I

Louise Fessard, Mediapart

The same applies to the media's submission to the poliJcal and media agenda, which generally forces journalists to cover the hoZest news. However, our respondents consider that it is someJmes necessary -and rewarding -to work on events that are not in the main agenda. For them, it is therefore a quesJon of breaking with the injuncJon to instantaneity:

[Working for Les Jours] I was really interested in this mul9-ar9cle format. To be able to dig into issues over several weeks, several months, and I func9on a bit like that, I have obsessions. I'm interested in all police issues. […] Since I've been here, almost two years, I've worked on four or five subjects, no more. It allows you to do the preliminary documenta9on, you're not a researcher but you can take the 9me to read books, to read the press that has already appeared to try to get up to speed on the subject, to go and see people with not just three specific ques9ons to ask them but a discussion to have with them on what would be interes9ng to do journalis9cally, What do they think of the way the media report their ac9vity, are there possible improvements in the journalis9c treatment of an issue, because there are subjects that have been lible or badly treated and that deserve to be revisited. […] It is quite sa9sfying intellectually.

Camille Polloni, Les Jours

Autonomy, Subjec6va6on, Proximity

To aZract competent journalists, media companies that invest in the margins of the field also understand that they have to rethink their internal organizaJon. For commiZed journalists, it is not so much a quesJon of joining a poliJcized media as of working in condiJons that suit them. These condiJons are open thought in opposiJon to the model of management and the editorial strategies in force within the mainstream media:

On

Camille Polloni, Les Jours

We send the ar9cles in the evening, in the morning Hervé Kempf reads them, he calls between 7 and 8 am for the last modifica9ons and at 8 or 9 am they are published. Hervé Kempf is editor-in-chief and director of the publica9on. In 2014, there were four journalists in Paris, and about twenty freelancers. […] I was free in Reporterre to write as I wanted. I was never rewriben. And therefore, I had total freedom to explain my percep9on of things.

Grégoire Souchay, Reporterre

In the choice of their subjects, sources and treatment, we therefore find among our interviewees an aspiraJon to autonomy that resonates strongly with that of the acJvists of the new social movements [START_REF] Ion | L'évoluJon des formes de l'engagement[END_REF][START_REF] Dupuis-Déri | L'altermondialisme à l'ombre du drapeau noir. L'anarchie en heritage[END_REF]. As a result, journalists and their sources are connected by 'elecJve affiniJes' [START_REF] Löwy | Le concept d'affinité élecJve chez Max Weber[END_REF]: autonomy as a poliJcal value resonates with the radical media's desire to free journalism of the straitjacket of the dominant press. Our journalists, driven by autonomy, do not feJshize objecJvity and instead leave more room for their subjecJvity [START_REF] Lemieux | La subjecJvité journalisJque[END_REF]. The following passages aZest of a desire to work on subjects that affect them personally. But also, to 'change the order of things':

Basically,

Grégoire Souchay, Reporterre

This logic of subjecJvity is embodied in the proximity between our interviewees and their acJvist sources. As for local press correspondents, commiZed journalists' idenJty is constructed in the opposite way to the discourse founding the professional autonomy of journalists. It is through the search for social proximity and not through the distance established between the editor, the sources and the readers that belonging to the profession is forged" (Bouron, 2015, p. 96). Thus, our journalist respondents seek to build relaJonships of trust with acJvists (Thiong-Kay, 2021). This is a way for them to neutralize the 'substanJalist' criJque that considers journalism as an enJty governed by universal rules and logics, despite the diversity of media [START_REF] Nollet | Croiser analyse des poliJques publiques et sociologie des médias: genèses et usages des concepts de mise a l'agenda et de construcJon des problèmes[END_REF]. Similarly, they rely heavily on counter-experJse, 'fact checking' and material such as amateur video content that acJvists post on their websites and social media profiles. Finally, through the parJcipatory dimension of their media, commiZed journalists fully involve acJvists and militants in their producJon process.

Toward a New Mythology of Professional Excellence?

Journalists deploy this range of pracJces for different reasons. First, they are generally more in tune with the acJvist percepJon of the issues they are working on. Second, they know that their first readers will be acJvists or sympatheJc audiences. Third, unlike their mainstream counterparts, they have liZle access to insJtuJonal sources. Fourth, they want to show, on the ground, that 'another journalism' is possible. This 'other journalism' is not just a quesJon of invesJgaJve subjects or ways of handling informaJon. It is claimed as a reacJon to the coverage by mainstream media, and to the way the laZer manage the social distance separaJng them from their interviewees. Journalists want to disJnguish themselves from their colleagues, in the sense of [START_REF] Bourdieu | La disJncJon: CriJque sociale du jugement[END_REF]:

There's a colleague and friend who's not super flexible we'll say, who's a bit more into the salons. Well, he's got a good background, but he's not always prepared to go and see people with dreadlocks and dogs, things like that, whereas I don't have any worries. Anyway, I don't have any problems with anyone, really: suits and 9es, djellabas, I talk to everyone.

Frédéric Scheiber, freelance photojournalist

This logic of disJncJon of 'another journalism' promoted by our interviewees aims also in occupying a beZer posiJon in the journalisJc field. As we wrote above, journalists as a social group are characterized by strong internal compeJJon. This compeJJve dimension produces orders of magnitude by valuing specific trajectories, social capital and professional pracJces.

As we have said earlier, through the press card allocaJon commission, the field of journalism in France provides itself with a validaJon mechanism (symbolic and effecJve) that insJtutes certain actors and marginalizes others. This exacerbated compeJJon between members of the journalisJc field may lead some of them to assume an explicitly 'rebellious' posiJon with regard to these legiJmacy grids. It may be a quesJon of making the non-respect of the journalisJc cursus honorum a reason for audacity, and therefore excellence, which turns the sJgma of illegiJmacy to an advantage. The case of Gaspard Glanz is parJcularly enlightening here as he encounters the beginning of his career:

I

Gaspard Glanz, Taranis News

This criJcism of the journalisJc cursus honorum is not without consequences for the autonomy to which the acJvists aspire or which they claim, and which we have described above. For Gaspard Glanz, it becomes 'criJque in acJon' through the creaJon of a media centered on social conflict. It is expressed in the strengthening of a do-it-yourself journalisJc idenJty, of the reporter as an entrepreneur of himself in reacJon to a highly compeJJve field. He conJnues:

And s9ll today it remains, you see.

Gaspard Glanz, Taranis News

Thus, a more autonomous and subjecJve journalisJc counter-ideal is emerging. The laZer is a reacJon to the structural transformaJon of the journalisJc field. Indeed, in France, certain specialJes are constantly at the center of newsrooms. This is notably the case of economic journalism. Other specialJes are increasingly successful. This is parJcularly true of pundits and commentators who in France are called "editorialists". Generally ideologically conservaJve, editorialists are reacJvaJng a French-style model of journalism, whose emphasis on commentary, as opposed to reporJng, reflects the weight of meta-discourse on current events transforming the news into pretexts for the expression of opinions (Neveu, 2001, p. 14).

Editorialists who are very present and influenJal in French newsrooms repel our respondents, who are aZached to the legiJmacy of invesJgaJve journalism and fieldwork. And it is open in opposiJon to the editorialist model that our journalists posiJon themselves, drawing the contours of their ideal vision of the profession. Jade Lindgaard, for example, talks about the lasJng influence on her of the "muckracker" type of journalist, who, at the beginning of the 20th century in the United States, worked as close as possible to the working condiJons in the abaZoirs and slums of ManhaZan:

It is much more pres9gious and valuable as a posi9on than to tell stories from an overhang, or whatever. You have to go for it. On the contrary, you have to go all the way. That's the only way to scratch, how can I put it, this straitjacket of discourse, of dominant representa9ons.

Jade Lindgaard, Mediapart

Gaspard Glanz, for his part, calls for a certain fairness of treatment. He considers that the actors of the poliJcal and social world do not quesJon the poliJcal and ideological aZachments of editorialists, unlike those who are designated as commiZed journalists:

I'm a bit 9red of answering this ques9on, but today when Jean-Pierre Raffarin, Aurélie Filippev, Gaspard Gantzer, are judged as journalists when they are people who have just finished a forty-year career in poli9cs without ever having done anything else, […] hardcore poli9cians, these people are journalists from one day to the next and they are not considered to be ac9vists? Well, in that case I don't think I am either.

[…] The more 9me goes by, the more journalism and poli9cs become intertwined, journalists become poli9cians, poli9cians become journalists, and the less sense this ques9on of militancy has. Today you have raw repor9ng, which is what I try to do, and you have analysts, commentators, they are two different jobs.

Gaspard Glanz, Taranis News

In return, French editorialists regularly target commiZed journalists as their adversaries. A tweet from editorialist Brice Couturier, which reads "I had the opportunity to denounce this Gaspard Glanz in 'Les MaJns de France Culture' as an extreme lep acJvist disguised as a journalist", is a case in point.

"Radical Journalist": A Label to be Neutralized

We understand that the commiZed or radical journalist is not just an idenJty assumed by our respondents. It is also a label or a sJgma [START_REF] Becker | Outsiders: Studies in the Sociology of Deviance[END_REF] aZached by some of their resolutely hosJle colleagues. In reality, lep-wing radicalism remains marginal within the French journalisJc field. The reasons for this are of different kinds. First of all, although they are commiZed, the journalists studied remain aZached to what generally founds the legiJmacy of their acJvity. As Jocelyne Arquembourg (2011) explains, journalists "stabilize readings of the facts, of a temporal structure, distribute roles between protagonists to whom they aZribute responsibiliJes" (p. 34). Their acJvity "implies homogenizing, organizing, arJculaJng and confronJng a quanJty of heterogeneous informaJon" (Arquembourg, 2011, p. 25). Thus " media objecJvity [is] the basis of their legiJmacy in a democraJc regime" (2011, p. 2). Our journalists, however poliJcized or criJcal, sJll adhere to this definiJon of the role of journalists. They deploy an arsenal of techniques that consJtute a parJcular "reading contract" (Véron, 1985): contextualizaJon of events, factual narraJon, gathering of all points of view, use of quotaJon as a way of distancing themselves from the object of invesJgaJon, explicaJon of external resources (such as militant websites), etc. The use of these techniques is a necessary condiJon for the expression of subjecJvity. The deployment of the former allows for the acceptance of the laZer. As Jade Lindgaard explains:

I worked for the first 9me on the Sivens dossier, saying to myself, we have to do something, but that's always the problem when you've missed the beginning of a story, as they say in journalism, when you haven't been on the subject since the beginning, you have to get back up to speed: o\en these are technical and complex dossiers. […] for me, our role as journalists is to be in a kind of accelerated research compared to the 9me of the research, but a research [all the same], and therefore a documentary work, of factualisa9on of the problem, that seems important to me.

Jade Lindgaard, Mediapart

Grégoire Souchay offers a more consistent analysis. He theorizes the complexity of the posiJon he tries to hold between demanding external and internal constraints. Thus, he combines the respect of a certain journalisJc deontology with a personal aspiraJon for poliJcal change:

There is an expression I use to sum up my posi9on, it is "the ass between two chairs" and it is the posi9on I have always adopted and that I have chosen to take because I am not comfortable in the posture of the ac9vist and I am not comfortable in the posture of journalism alone. I need to combine commitment with professional work, without being militant, but without being neutral. This is my posi9on. But I am not an ac9vist in the sense that when I work, it is a profession. And I apply professional techniques.

Grégoire Souchay, Reporterre

CriJcized for belonging to radical media, and someJmes for their acJvism supposedly disguised as journalism, our respondents adjust their discourse. This adjustment is designed to ward off criJcism from their opponents in the media. But it is also formulated to resist other transformaJons of the media sphere. In this respect, we have already menJoned the presence in social movements of a type of media acJvism who someJmes defines itself as 'self-media' (automedia). This kind of poliJcal and communicaJonal acJvism (Thiong-Kay, 2020) also contributes to widening the borders and modifying the structure of the journalisJc field. If we consider the mediaJzaJon of certain struggles, a range of statuses and idenJJes coexist: selfmedia, ciJzen reporters, independent journalists without a press card, commiZed journalists with a press card, amateur contributors to parJcipatory media, etc. However, when compared to these "associate-rivals" [START_REF] Neveu | Médias, mouvements sociaux, espaces publics, Réseaux n° 98[END_REF], our respondents wish to differenJate themselves: In journalism, recourse to ethics funcJons as a modality of self-insJtuJon, because the profession is subject to compeJJon and criJcism from the social worlds around it [START_REF] Ruellan | Nous journalistes. Déontologie et idenJté[END_REF]. For journalists, invoking ethics makes it possible to establish orders of magnitude intended to specify -or even confine -the roles of each person, but above all to jusJfy the relevance of their own work. While Grégoire Souchay recognizes the funcJonal complementarity of commiZed journalism and acJvism, such a consideraJon allows him to reaffirm the specificity and contribuJon of his own professional acJvity. According to him, being a professional journalist implies not only reporJng what is happening 'on the ground', but also knowing how to use a whole conceptual and analyJcal panoply to qualify and explain the facts. Such a posiJon relegates the self-media to its primary status of a source or an actor directly implicated in the events. But it can also consist in downgrading it symbolically, by reducing it to a militant ersatz of the editorialists who dominate large news television channels. Thus, the funcJon of journalism, even if it is commiZed, consists in reporJng the facts, but above all in contribuJng to their deciphering, qualificaJon and reinserJon in a given context.

I
To conclude, we must specify that the register of commiZed journalism someJmes turns against the interviewees themselves. This is a backlash against the current structure of the journalisJc field and its strong polarizaJon, as menJoned above. Thus, several of our interviewees menJoned the difficulty of accessing official sources. They also tesJfy to an inability to access certain demonstraJons and thus do their job, because they are blocked by police. The Yellow Vests movement and a recent drap law in France have shed a harsh light on these pracJces, which even commiZed journalists find very difficult to cope with.

Clearly, they were the ones who had the first informa9on.

[…] The journalists from La Dépêche, […] they had a greater capacity to access official sources, so we heard them more. So, you could hear the prefect, you couldn hear the pro-dam farmers, the FNSEA . I didn't have access to these sources.

[…] For the zadists I was Reporterre, whereas I saw myself as a journalist and that's why at the beginning of September when there were police blockades, the head of the gendarmerie told me a\er passing a first blockade: "I said that Reporterre didn't pass" […] The big problem I had with the cops was the blockades. According to them, I wasn't a journalist, so I didn't go through the roadblocks.

Grégoire Souchay, Reporterre

These pracJces of the public authoriJes and the police forces prevent journalists from collecJng all points of view. They are the translaJon of two public representaJons. Firstly, they inform us about a 'funcJonalist' reading of the journalisJc field, which would thus be a simple copy of the poliJcal field. Secondly, these pracJces correspond to a 'determinisJc' reading of the journalist with whom the official source or the representaJve of the police forces is confronted. The journalist is thus assigned to his press card, his specializaJon, his supposed poliJcal convicJons and the media to which he belongs, as if he were a prisoner. In both cases, these representaJons favor a mechanical idenJficaJon of commiZed journalists to militant sources. This can be observed empirically in the field.

Indeed, Grégoire Souchay has to find a way into his field of invesJgaJon in Sivens. He then takes the same route as the acJvists to bypass the police blockade and reach the ZAD. Consequently, the forced division of journalisJc labor also has very physical causes in the media coverage of a controversy. It reinforces the rouJnizaJon of relaJons between journalists and a certain type of source, someJmes confronted with the same adversity. In this sense, the quesJon of journalisJc access to the plurality of actors in a conflict has a performaJve dimension. It establishes entry rights and refusals that influence not only the media's treatment of the conflict on D-day, but also affects that of the following day and the day aper.

The problem of access to the field, which is closely linked to the polarizaJon of the journalisJc field, has a major impact on interacJons on the ground. It therefore condiJons the content of the journalisJc producJon.

So I went through the forest and I made the same journeys as the zadists.

And so I reinforced the image that I was a zadist. It's like in demonstra9ons, there are guys wearing balaclavas who aback the cops, so there are people who wear scarves to protect themselves, so they are seen as balaclavas. [] Basically, you are just an enemy. On the day of the burials (protestors burying themselves in the Sivens forest), I found myself stuck with the buried guys, surrounded by cordons of cops for three hours.

Grégoire Souchay, Reporterre

In order to do their work in decent condiJons, professionals need to neutralize, at least in part, the label of 'commiZed journalism', which would imply a bias. In these condiJons, loyalty to journalisJc ethics is a reason for resilience in the minds of our journalists. This allows them to maintain intellectual rigor and professional jusJficaJon, which are expressed through their aZachment to invesJgaJon, to the descripJon of public policies or to the study of local configuraJons. For example, when asked about her view of the 'Grands Projets InuJles et Imposés', in general, Camille Polloni refuses to pass judgment from above without having access to the data on each of the projects fought by the militants:

I'm not necessarily comfortable answering that, actually. I don't always know the details of the project and I don't always have the tools to judge their relevance. I read the arguments on both sides. What interests me more is the mobiliza9ons against and the way they are structured, the journey of the people who come to join the ZADs, more than the project itself. But the project itself I have never been led to analyze it in detail to form an absolutely defini9ve opinion on it.

Camille Polloni, Les Jours

Conclusion

In this chapter, we have examined lep-wing radical journalism in France. The commitment of journalists can consist in consciously parJcipaJng in the construcJon of public problems. From an opposiJonal perspecJve [START_REF] Negt | L'espace public opposiJonnel[END_REF], this commitment aims to fight against the pejoraJve stereotypes that target acJvists. It helps overcome the perceived dysfuncJons of the journalisJc field. In this sense, the commitment of our respondents seeks to bring about a journalisJc counter-field, in which professionals would accept their share of subjecJvity and construct new moJves for journalisJc excellence. However, it should be pointed out that for the actors, this perspecJve must be carried out in permanent tension with different elements: the normaJve framework of the profession, the compeJJon of new informaJon producers and the adversity of the poliJcal context. The noJon of the 'commiZed' or 'radical' journalist is therefore operaJve from an empirical -analyJcal -point of view. However, its social and scienJfic use must be rigorously apprehended.

  had been rewriben, you could feel the weight (he insists on this word) of rou9nes and heaviness that there was to write[…] and then … the self-censorship, even direct censorship. There was really this thing where you said wow[…]. I really don't want to be there.

	Grégoire Souchay, Reporterre
	The facts that are reported by the regional press, generally we see quite
	quickly that it is told through the police version, and there is very lible room
	for another version because it is told the very next day, […] I understand, I
	don't necessarily know how to meet the family, etc. […] I understand, I
	know a lot of journalists from La Provence (main newspaper in the region
	of Marseille), for example, there's an emergency, they have to fill in the

had done a month's internship at La Dépêche du Midi in Toulouse, I didn't like it

[…]

As a local intern during the summer, it was really disgus9ng and the lible I had done (he blows his nose), it paper for the next day, etc. […] They're the ones who have to do it.

[…]

  the web, there is Rue89, which is in the process of collapsing, there is Mediapart, which is doing very, very well, there is Les Jours, which is also star9ng to do well a\er a year, and Basta! […] In a few years, openings have been created everywhere. With organiza9ons that are also flexible and interes9ng. Here, in the way we work, we are 26 employees, but not like in a very hierarchical daily newspaper with a head of department on every floor. I have two editors-in-chief, and it's one or the other who proofreads my papers, who gives me indica9ons on what I could do, who suggests things. It's also a more pleasant way of organizing work.

  the scale of humanity, but it's my own history. I won't live long but on my scale it's interes9ng. […] In fact, it's super ego9s9cal, because I do things that interest me. And I'm not good at it because I don't think about the subjects that interest "them", the press. I do things that interest me. I do my own journalism, in fact. Europacity if it is built, it's really a problem and well 1), Mediapart allowed me to do it, 2) it allowed me to do ar9cles that were different from the ar9cles published by the newspapers or media with which we compare ourselves, so that means the mainstream media Le Monde, Les Echos, Le Figaro for example, and 3) it allowed me to, how shall I put it, to gain a par9cular audience who came to read it. You see, it's this rela9onship that's a bit difficult, because an ordinary journalist doesn't have to have an impact somewhere. You make the news, and it's pure news. And if it has an impact, it's almost suspect. So there is this thing, this posi9oning that is a bit complicated.

	Frédéric Scheiber, freelance photojournalist
	I insist on this: posing as a journalist on ecology means that yes, it is a
	commitment [… I insist on this: posing as a journalist on ecology means
	that yes, it's a commitment […] i.e. the Notre-Dame-des-Landes airport
	must not be built,

there are things that interest me, a ZAD is opening up not far away, I'm interested in an9-capitalists, I want to know. For me it's part of history! It's important, there are things that are created, even if they are epiphenomena on Jade Lindgaard, Mediapart Journalism […] I have the impression that it is the means by which I am most useful for advancing "The Cause", that is to say that it is my lever. Some people are going to be trade unionists in their company, some are going to write revolu9onary pamphlets like the Invisible Commibee, some are going to be involved in a party or take up posi9ons of power.

[…]

Where I am effec9ve and useful is here.

[…] I didn't write an ar9cle on Sivens to abract militants, but if it did, then it was useful.