domain: scco shs shs.psy

text:
Hadrien Ceyte
email: hadrien.ceyte@univ-lorraine.fr

Jean-Marie Beis

Gwenaelle Ceyte

Sébastien Caudron

Perceptual post-effects of left neck muscle vibration with visuo-haptic feedback in healthy individuals: a potential approach for treating spatial neglect

Keywords: neck muscle vibration, visuo-haptic feedback, combined intervention, straightahead, intermodal calibration, neglect rehabilitation

Among the techniques used to reduce spatial neglect's symptoms, left neck muscle vibration (NMV) is alluring because it does not require the patient's attentional co-operation. The aim of this study was to determine the type of NMV-associated feedback that induced the most intense and longest-lasting egocentric post-effects. Eighty-seven healthy individuals were randomly assigned to four intervention groups: "neck muscle vibration, blindfolded" (NMV), "neck muscle vibration with vision" (NMV+V), "neck muscle vibration and visual fingerpointing" (NMV+P), and "visual finger-pointing" (P). An eyes-closed finger-pointing subjective straight-ahead (SSA) test was carried out before the intervention, immediately afterwards, and 30 minutes afterwards. The results showed that only the NMV+P intervention induced a lasting leftward bias of SSA. In addition, the deviation reported in this intervention group differed significantly from those observed in the other interventions. The combination of visuo-haptic feedback and neck-somatosensory stimulation may enable a full, lasting intermodal recalibration, which could be potentiated by the attention level engaged during voluntary pointing. These outcomes highlighted that the NMV technique could easily integrate into routine occupational therapy sessions for treating various aspects of neglect disorders.

Introduction

Unilateral spatial neglect (USN) is a perplexing neuropsychological syndrome that affects several domains of spatial cognition and also impacts functional domains [START_REF] Verdon | Neuroanatomy of hemispatial neglect and its functional components: a study using voxel-based lesionsymptom mapping[END_REF]. The damaged brain networks (mostly in the right hemisphere) appear to affect the whole system of spatial coordinates relative to the patient's body [START_REF] Vallar | A frontoparietal system for computing the egocentric spatial frame of reference in humans[END_REF]. More specifically, these patients fail to detect and respond to stimuli located contralaterally to the hemispheric lesion. Moreover, many studies of egocentric perception in people with left USN have shown that the subjective straight-ahead (SSA) is deviated to the right [START_REF] Chokron | Variations of the egocentric reference among normal subjects and a patient with unilateral neglect[END_REF][START_REF] Richard | Straight ahead in spatial neglect Evidence that space is shifted, not rotated[END_REF]. This impairment can contribute to longterm disability.

Several rehabilitation approaches have been developed to reduce spatial neglect.

Conventionally, these approaches can be categorized as "top-down" (based on the patient being consciousness of their impairment) or "bottom-up" (based on sensory manipulation).

"Bottom-up" sensory stimulation is alluring because it does not require much attentional cooperation by the patient; this is notably an important advantage in early-phase rehabilitation [START_REF] Johannsen | Lasting amelioration of spatial neglect by treatment with neck muscle vibration even without concurrent training[END_REF][START_REF] Azouvi | Rehabilitation of unilateral neglect: Evidence-based medicine[END_REF][START_REF] Kerkhoff | Modulation and rehabilitation of spatial neglect by sensory stimulation[END_REF].

Neck muscle vibration (NMV) has been described by Saevarsson et al. [START_REF] Saevarsson | Designing Rehabilitation Programs for Neglect: Could 2 Be More Than 1+1?[END_REF] as a passive-restorative intervention technique and has shown promising results in patients with left USN. This proprioceptive stimulation (application of 80-100 Hz vibration to the belly of a muscle) generates muscle spindle and Ia afferent fiber responses that are interpreted centrally as a change in muscle length [START_REF] Goodwin | The contribution of muscle afferents to kinaesthesia shown by vibration induced illusions of movement and by the effects of paralysing joint afferents[END_REF]. In healthy individuals, left-side NMV affects the whole egocentric coordinate system and induces (i) a rightward illusory motion of a stationary visual target, and (ii) a leftward deviation of the visually estimated SSA [START_REF] Biguer | Neck muscle vibration modifies the representation of visual motion and direction in man[END_REF][START_REF] Ceyte | Effects of neck muscles vibration on the perception of the head and trunk midline position[END_REF][START_REF] Karnath | The interactive contribution of neck muscle proprioception and vestibular stimulation to subjective "straight ahead" orientation in man[END_REF]. These perceptual egocentric effects are also observed in patients with various perceptive or sensorimotor impairments [START_REF] Strupp | Perceptual and oculomotor effects of neck muscle vibration in vestibular neuritis. Ipsilateral somatosensory substitution of vestibular function[END_REF][START_REF] Karnath | Subjective body orientation in neglect and the interactive contribution of neck muscle proprioception and vestibular stimulation[END_REF] and they are reportedly more intense in patients with USN [START_REF] Karnath | Subjective body orientation in neglect and the interactive contribution of neck muscle proprioception and vestibular stimulation[END_REF] and in patients with spatial cognition disorders [START_REF] Strupp | Perceptual and oculomotor effects of neck muscle vibration in vestibular neuritis. Ipsilateral somatosensory substitution of vestibular function[END_REF] than in healthy individuals. In patients with left USN, the effects of left-side NMV might lead to improved sensorimotor coordination.

Moreover, some studies have found that left-side NMV produces a lasting reduction in the symptom of spatial neglect [START_REF] Johannsen | Lasting amelioration of spatial neglect by treatment with neck muscle vibration even without concurrent training[END_REF][START_REF] Schindler | Neck muscle vibration induces lasting recovery in spatial neglect[END_REF][START_REF] Ceyte | Lasting improvements in left spatial neglect following a protocol combining neck-muscle vibration and voluntary arm movements: a case-study[END_REF][START_REF] Vallar | Improvement of left visuo-spatial hemineglect by left-sided transcutaneous electrical stimulation[END_REF].

Biguer et al [START_REF] Biguer | Neck muscle vibration modifies the representation of visual motion and direction in man[END_REF] suggested that perceptual effects are optimized when the NMV technique is applied in darkness. In the literature, however, most studies of NMV-based rehabilitation for USN maintained normal ambient light conditions, and patients were allowed or instructed to keep their eyes open [START_REF] Johannsen | Lasting amelioration of spatial neglect by treatment with neck muscle vibration even without concurrent training[END_REF][START_REF] Schindler | Neck muscle vibration induces lasting recovery in spatial neglect[END_REF][START_REF] Vallar | Improvement of left visuo-spatial hemineglect by left-sided transcutaneous electrical stimulation[END_REF]. This raises the important question of whether or not the visual feedback-context during NMV influences the magnitude of any induced post-effects. Furthermore, the passive nature of NMV means that it can be easily combined with other passive techniques (e.g. vestibular, optokinetic or transcutaneous electrical nerve stimulation), or with active techniques (e.g. prism adaptation and visual exploration training).

Likewise, several studies have reported that post-effects in patients with USN were stronger and longer lasting when treatments with converging effects were combined [START_REF] Kerkhoff | Modulation and rehabilitation of spatial neglect by sensory stimulation[END_REF][START_REF] Saevarsson | Designing Rehabilitation Programs for Neglect: Could 2 Be More Than 1+1?[END_REF][START_REF] Karnath | Subjective body orientation in neglect and the interactive contribution of neck muscle proprioception and vestibular stimulation[END_REF][START_REF] Luauté | Visuo-spatial neglect: A systematic review of current interventions and their effectiveness[END_REF][START_REF] Pitteri | Is two better than one? Limb activation treatment combined with contralesional arm vibration to ameliorate signs of left neglect[END_REF].

In some "top-down" interventions combined with NMV, patients have to be strongly aware of their spatial impairment so that they can compensate actively for the resulting spatial bias [START_REF] Kerkhoff | Modulation and rehabilitation of spatial neglect by sensory stimulation[END_REF][START_REF] Schindler | Convergent and divergent effects of neck proprioceptive and visual motion stimulation on visual space processing in neglect[END_REF]. This requires patients with left USN to engage substantial attentional resources.

However, an implicit awareness of spatial bias during performance can be easily induced by (for instance) asking the patient to reach specific visual targets in their peripersonal space with their arm. Awareness of bias between the arm's final position and the target's position would be facilitated by visual and haptic feedback. In a preliminary study [START_REF] Ceyte | Lasting improvements in left spatial neglect following a protocol combining neck-muscle vibration and voluntary arm movements: a case-study[END_REF], we described the effectiveness of treatment based on left-side NMV and visuo-haptic feedback in an patient with left USN. A generalized, lasting (24-hour) reduction in neglect symptoms (including better wheelchair navigation) was observed. However, it was not clear whether the treatment effect was due the repeated reaching movements, the left-side NMV, and/or the visuo-haptic feedback. The lack of theoretical knowledge on the influence of sensory feedback during the application of NMV has led to a diversity of environmental contexts in the application of this technique. However, it is not known which of these environments provides the strongest or longest-lasting post-effects.

The present study's primary objective was to describe and compare the perceptual post-effects induced by left-side NMV (i.e. deviation of the SSA immediately after stimulation and its persistence at 30 minutes) under visual or visuo-haptic contexts in a large group of healthy individuals. Thereafter, the objective will be to propose the sensory feedback context that was associated with the greatest post-effects and that might be of relevance in patients with left USN. Our hypothesis was that visuo-haptic feedback-context associated with the NMV technique would induce the most promising post-effects.

Materials and methods

Participants

Eighty-seven right-handed, right dominant eye, normal-sighted, healthy young adults (including 39 women, age: 21.4±2.2, respectively: mean±standard deviation) were recruited in the study. They were randomly assigned to four independent groups (random drow computed with a Matlab script), each of which corresponded to a single type of intervention: a "neck muscle vibration, blindfolded" group (NMV: n=21, including 5 women; 21.3±2.8); a "neck muscle vibration with vision" group (NMV+V: n=22, including 6 women; 20.7±1.7); a "neck muscle vibration with visual finger-pointing" group (NMV+P: n=24, including 15 women; 22.2±2.3); and a "visual finger-pointing" group (P: n=20, including 13 women; 20.8±1.1). None of the participants had a history of central nervous system disease or psychiatric, neurological, ocular, oculomotor or vestibular disorders. None of the participants complained of dizziness or vertigo during the study. The study was carried out in agreement with the French and European legislation and the Declaration of Helsinki.

Experimental devices

The participant sat comfortably in a chair in front of a wooden device placed on a table. He/she placed their chin on a height-adjustable chinrest, so that the head remained vertically aligned with their trunk throughout the experiment. Five light targets (LEDs, 0.

Experimental design

• The SSA task and the test sessions During a test session, the participant's chin was always placed on the chinrest in order to align the participant's straight-ahead with the zero-degree direction of the wooden device. The right index finger positioned at the base of the chinrest (in front of the sternum). The blindfolded participant was asked to indicate their SSA with the right index as quickly as possible. The final index position had to be maintained for at least 2 seconds, before a return to the starting position at the base of the chinrest. Twenty pointing movements were carried out in each test session. Three test sessions were performed: before the intervention (Pre), immediately afterwards (Post0), and 30 minutes afterwards (Post30).

• Procedure for each intervention Each participant was equipped with a vibratory stimulator on each upper trapezius muscle, and the head position was maintained on the chinrest. In the NMV, NMV+V and NMV+P groups, only the left-side vibrator was activated (vibration frequency: 100 Hz; vibration amplitude: 300 µm) throughout the 15-minute session; the right vibrator was never activated.

In the NMV group, the blindfolded participants did not have to perform any specific activities during the left-side proprioceptive stimulation of the neck, other than an informal discussion with the investigator. In the NMV+V group, the stimulation conditions were the same as in the NMV group, except that participants were not blindfolded and could keep their eyes open.

In the NMV+P group, the participants were asked to point as quickly as possible at one of the After the target had been indicated, the participant had to return the index finger to the starting position at a natural speed. Five blocks of 50 successive pointing trials were performed. Each pointing direction was presented 10 times, in random order. Each block lasted for approximatively 2.5 minutes, and there was a 30-seconds rest period between successive blocks. In the P group, the participants performed the same finger-pointing task as the NMV+P group but in the absence of any vibratory stimulation; this enabled us to check the possible training effect of repeated finger-pointing on post-effects in the NMV+P group.

Data reduction and statistical analysis

As previously mentioned, the participant's objective straight-ahead was perfectly aligned with the zero-degree direction of the wooden device. For each trial, the direction indicated by the blindfolded participant with their index finger (corresponding to their SSA) was visually measured thanks to a circular degree scale located on the edge of the wooden device. The precision of this measurement was ±0.5 degrees. Leftward and rightward errors were assigned negative and positive values, respectively.

For each test session, quantitative data were expressed as the medians (M) [interquartile range (IQR)]. Under all the four stimulation conditions, the data were not normally distributed (Shapiro-Wilk test, ps<0.05) and heterogeneity of group-variances occurred (Levene's test, ps<0.05). Therefore, non-parametric statistical analyses were computed. For the study participants as a whole and then for each group, Friedman's test was used to analyze the effect of the period (Pre; Post0; Post30) on the median SSA values. When Friedman's test was significant (with an alpha level of 0.05), post-hoc Wilcoxon's signedrank tests (with a Bonferroni-adjusted alpha level of 0.017) was used to assess pairwise comparisons between each of the three periods (Pre; Post0; Post30). Kruskal-Wallis tests were used to compare the intervention groups. Firstly, a Kruskal-Wallis test was performed on the SSA's sums of ranks in each group (NMV; NMV+V; NMV+P and P) at Pre, in order to ensure that the SSA values were equivalent at baseline level. Secondly, the effect of the intervention was computed for each participant at Post0 and at Post30 respectively by subtracting the baseline level from mean SSA after intervention (i.e. respectively mean SSA at Post0 minus mean SSA at Pre and mean SSA at Post30 minus mean SSA at Pre). Therefore, leftward and rightward SSA variations were assigned negative and positive difference values, respectively. In order to analyze the effect of the intervention type on SSA variations, Kruskal-Wallis tests were performed on the SSA Post-Pre differences' sums of ranks in each group (NMV; NMV+V; NMV+P and P), i.e. Post0-Pre and Post30-Pre SSA's differences.

When Kruskal-Wallis test was significant (with an alpha level of 0.05), post-hoc pairwise comparisons between groups were computed and p-value adjusted with the Steel-Dwass-Critchlow-Fligner (SDCF) method [START_REF] Critchlow | On distribution-free multiple comparisons in the oneway analysis of variance[END_REF].

Results

The For the study participants as a whole (i.e. independently of any group effect), the median SSA was significantly modified by the intervention, as indicated by the significant

Discussion

The present study's objectives were to compare the egocentric post-effects induced by left-side NMV under various sensory feedback contexts in a large group of healthy individuals and to select the context that induced the most intense, persistent perceptual posteffects. Although the environment in which NMV is applied has never been investigated per se, it has a fundamental impact on the technique's effectiveness. This impact is important with regard to improving the rehabilitation of spatial neglect. Firstly, our results showed that the SSA in healthy right-handed participants is deviated slightly rightwards [START_REF] Chokron | Variations of the egocentric reference among normal subjects and a patient with unilateral neglect[END_REF][START_REF] Chokron | Visual, proprioceptive and tactile performance in left neglect patients[END_REF]. Secondly, our study's main finding is that only the "NMV with visuo-haptic feedback" intervention induced a leftward deviation of the egocentric frame of reference for at least 30 minutes. These perceptual post-effects were not attributable to the visuomotor training induced by repeated pointing. Lastly, and regardless of whether or not visual feedback was present during left-side NMV, we did not see a post-effect on the SSA in the absence of haptic feedback.

A neural mechanism favorable to an intermodal updating process

Although the study's psychophysical data cannot provide direct evidence of the neural mechanisms induced by the "NMV with visuo-haptic feedback" intervention, they (and the literature data in the field of neurophysiology) enable us to form a number of hypotheses. The effects induced seem relatively similar to those induced by other "bottom-up" interventions (e.g. vestibular stimulation) and that might result from neuroplastic changes in the contralateral (right) hemisphere. However, several studies [START_REF] Vallar | Improvement of left visuo-spatial hemineglect by left-sided transcutaneous electrical stimulation[END_REF][START_REF] Bottini | Cerebral representations for egocentric space Functional-anatomical evidence from caloric vestibular stimulation and neck vibration[END_REF] have shown that somatosensory stimulation of the neck not only activates the contralateral hemisphere nonspecifically but also activates many vestibular neurons in the parieto-insular vestibular cortex.

As described in the monkey by Grüsser et al. [START_REF] Grüsser | Localization and responses of neurones in the parieto-insular vestibular cortex of awake monkeys (Macaca fascicularis)[END_REF][START_REF] Grüsser | Vestibular neurones in the parieto-insular cortex of monkeys (Macaca fascicularis): visual and neck receptor responses[END_REF], these neurons are known to (i) have bilateral receptive fields, (ii) participate in the multisensory integration of vestibular, optokinetic and somatosensory inputs, and (iii) contribute to the construction and updating of internal representations of head and body positions in space [START_REF] Vallar | Improvement of left visuo-spatial hemineglect by left-sided transcutaneous electrical stimulation[END_REF]. Vallar et al. [START_REF] Vallar | Improvement of left visuo-spatial hemineglect by left-sided transcutaneous electrical stimulation[END_REF] hypothesized that somatosensory stimulation of the neck has specific effects (possibly mediated by multimodal vestibular units) and may account for the reduction in neglect.

Indeed, the left-side NMV might counteract the (rightward) ipsilesional distortion of egocentric representations caused by (right) parietal lesions in patients with left USN. To do so, the vibratory stimulation might activate right hemisphere units with bilateral receptive fields. This neuronal process might underlie the perceptual effects observed in the present study.

A number of psychophysiological studies have corroborated the neurophysiological outcomes described above. Indeed, several studies have shown that the unilateral NMV provokes postural adjustments [START_REF] Jamal | The effects of neck muscle vibration on postural orientation and spatial perception: A systematic review[END_REF][START_REF] Popov | Postural responses to vibration of neck muscles in patients with unilateral vestibular lesions[END_REF], illusory body motion [START_REF] Goodwin | The contribution of muscle afferents to kinaesthesia shown by vibration induced illusions of movement and by the effects of paralysing joint afferents[END_REF][START_REF] Lackner | Some proprioceptive influences on the perceptual representation of body shape and orientation[END_REF], and illusory motion of a bright stationary target in an otherwise dark environment [START_REF] Biguer | Neck muscle vibration modifies the representation of visual motion and direction in man[END_REF][START_REF] Lackner | Changes in apparent body orientation and sensory localization induced by vibration of postural muscles: Vibratory myesthetic illusions[END_REF]. From a mechanical point of view, Strupp et al. [START_REF] Strupp | Perceptual and oculomotor effects of neck muscle vibration in vestibular neuritis. Ipsilateral somatosensory substitution of vestibular function[END_REF] showed horizontal eye deviation towards the vibrated side in patients with subacute unilateral vestibular lesions and also in healthy-participants, i.e. shifting the gaze off a stationary target so that it seems to move to the opposite side; this is congruent with the apparent target motion usually reported by participants. Lackner and Levine [START_REF] Lackner | Changes in apparent body orientation and sensory localization induced by vibration of postural muscles: Vibratory myesthetic illusions[END_REF] referred to this phenomenon as a "propriogyral illusion", in order to emphasize its similarity to the "oculogyral illusion" elicited by vestibular stimulation [START_REF] Graybiel | The oculo-gyral illusion; a form of apparent motion which may be observed following stimulation of the semicircular canals[END_REF]. Here, the visual component of illusory motion reflects the effect of proprioceptive stimulation of the neck on the neural representation of the direction of gaze, and highlights the importance of neck input for eye movement control [START_REF] Bronstein | Plastic Changes in the Human Cervicoocular Reflex[END_REF][START_REF] Popov | Visual and oculomotor responses induced by neck vibration in normal subjects and labyrinthine-defective patients[END_REF]. Moreover, Strupp et al. [START_REF] Strupp | Perceptual and oculomotor effects of neck muscle vibration in vestibular neuritis. Ipsilateral somatosensory substitution of vestibular function[END_REF] proposed that the effect of unilateral NMV on SSA resulted from changes in eye position and was possibly mediated by the cervico-ocular reflex. These observations suggest that the neck region has a strong influence on the central representation of body orientation [START_REF] Vallar | Improvement of left visuo-spatial hemineglect by left-sided transcutaneous electrical stimulation[END_REF][START_REF] Grüsser | Cortical representation of head-in-space movement and some psychophysical experiments on head movement[END_REF]. Thus, the somatosensory stimulation induced by unilateral NMV may have specific directional effects (deviation towards the stimulated side) on egocentric coordinates. Although this mechanism might account for the temporary improvement in neglect, it does not explain the persistence of post-effects with visuo-haptic feedback observed in the present study.

Overall, reaching a visual target requires proper sensory matching between the somatosensory afferences from the effector arm and visual afferences from the actual target position. Furthermore, asking someone to focus their attention on the visual target to reach, would activate the visual pathways, the posterior parietal areas and the motor cortices [START_REF] Wurtz | Brain Mechanisms of Visual Attention[END_REF][START_REF] Conte | Attention influences the excitability of cortical motor areas in healthy humans[END_REF].

According to Conte et al. [START_REF] Conte | Attention influences the excitability of cortical motor areas in healthy humans[END_REF], the attentional processes engaged would potentiate mechanisms of short-term plasticity in cortical motor areas. Thereby, two independent mechanisms, possibly complementary, might be the cause of the persistence of the post-effect.

Firstly, the combination of visuo-haptic and neck-somatosensory stimulation could produce a lasting multisensorial updating process, i.e. complete intermodal recalibration. Secondly, the activation of attentional processes for the fulfilment of a sensory-motor task coupled to necksomatosensory stimulation could potentiate the motor cortices' plasticity. It is also quite conceivable, through the neurophysiological and psychophysiological mechanisms presented above, that the addition of neck-somatosensory stimulation could activate a process of persistent intermodal recalibration which would be potentiated by the sensory-motor attention engaged by the individual during voluntary action.

Possible applications of NMV-based interventions in rehabilitation

Firstly, the non-invasive application of the neck vibrator device is an easy procedure to implement. However, the main advantage of the NMV technique is the weak requirement for attentional co-operation by the patient while obtaining significant changes in their representation of egocentric space [START_REF] Kerkhoff | Modulation and rehabilitation of spatial neglect by sensory stimulation[END_REF]. The NMV technique is known as a passive restorative intervention but it may facilitate the visuospatial detection of objects -a process that usually requires significant attentional resources in patients with USN -thanks to its incidental effect on eye position [START_REF] Strupp | Perceptual and oculomotor effects of neck muscle vibration in vestibular neuritis. Ipsilateral somatosensory substitution of vestibular function[END_REF]. The present study corroborated the post-effects induced by combining left-side NMV with visual finger-pointing, as shown previously in an patient with left USN [START_REF] Ceyte | Lasting improvements in left spatial neglect following a protocol combining neck-muscle vibration and voluntary arm movements: a case-study[END_REF]. These results suggest that sensorimotor self-activation during left neck-proprioceptive stimulation will obtain the most promising post-effects. The transfer of this intervention directly into rehabilitation sessions might further increase the intensity and/or the impact of the outcomes. To do so, reaching towards a target is very much like the motor activities executed during standard rehabilitation sessions in patients with USN. Consequently, the addition of the NMV technique during occupational therapeutic sessions might readily enhance the therapeutic effects obtained by standard treatments, without needing to add additional, specific sessions as is sometimes suggested [START_REF] Kamada | Effects of 5 minutes of neck-muscle vibration immediately before occupational therapy on unilateral spatial neglect[END_REF].

Lastly, the "NMV with visuo-haptic feedback" intervention involved voluntary movements with the participant's dominant (right) arm in the present study. Therefore, this intervention is not similar to the so-called "limb activation" therapy used in the cases of neglect [START_REF] Robertson | Spatiomotor cueing in unilateral left neglect: three case studies of its therapeutic effects[END_REF][START_REF] Robertson | Rehabilitation of unilateral neglect: improving function by contralesional limb activation[END_REF]. In limb activation therapy, the patient with left USN is asked to perform voluntary movements in peripersonal space with their contralesional limb (the left one).

Robertson et al.'s report on these patients showed that moving the left limb in the neglected hemispace significantly reduced the degree of neglect [START_REF] Robertson | One hand is better than two: Motor extinction of left hand advantage in unilateral neglect[END_REF]. However, they also found that adding right limb movements in the neglected hemispace abolished or at least dramatically limited this benefit. In order to explain this "motor extinction" effect, Robertson et al.

suggested that "the right hand movements are more perceptually salient than those of the left hand, and hence "overshadow" the latter."

In the light of these outcomes and with a view to applying the NMV technique during occupational therapeutic sessions for USN, it might be advantageous to combine the NMV technique with limb-activation. However, in the early stages of neglect, many patients have immense difficulty moving the limbs of the neglected hemi-body [START_REF] Kerkhoff | Modulation and rehabilitation of spatial neglect by sensory stimulation[END_REF]. Therefore, as a first step and in order to maintain motivation levels, these patients should be allowed to move only their ipsilateral (right) arm in their peripersonal space. The therapist will check the patient never moves both arms at the same time in the neglected hemispace. At present, there are no literature data on whether ipsilateral arm movements in the neglected hemispace induce less benefits than contralesional arm movements in the neglected hemispace; this topic warrants evaluation.

Conclusion

Our present results clearly emphasize the potential of combing visuo-haptic feedback with left-side NMV to optimize a lasting leftward egocentric deviation. The transfer of this combined intervention is realistic and relevant into rehabilitation sessions for various reasons.

The first is that the NMV technique is an easy procedure to implement and painless, and requires a weak attentional co-operation by the patient. The second is that repeated reaching movements are very much like the motor activities executed during standard rehabilitation sessions in patients with USN. Consequently, our findings argue in favor of including the NMV technique in rehabilitation sessions for patients with USN.

Figure legend

  3 cm in diameter) were placed at specific lateral locations 50 cm in front of the participant: straightahead, 10 degrees to the left and to the right, and 20 degrees to the left and to the right (i.e. -10 deg, -20 deg, 0 deg, 10 deg, and 20 deg). Vibratory stimulators (VB115, Vibrasens ® , Techno Concept, Manosque, France) were fixed bilaterally over the belly of the upper trapezius muscle and fastened with straps.

  five light targets (-10 deg, -20 deg, 0 deg, 10 deg, and 20 deg) with the right index finger.

  median [IQR] values of the SSA at Pre, Post0 and Post30 periods are depicted as a boxplot in Figure. *************** Please insert Figure here *************** In the Pre period, the median SSA did not differ significantly [H(3, N=87)=2.38; p=0.50] when comparing the NMV group (MNMV/Pre=+3.45 deg; IQRNMV/Pre=3.85), the NMV+V group (MNMV+V/Pre=+2.88 deg; IQRNMV+V/Pre=5.10), the NMV+P group (MNMV+P/Pre=+3.63 deg; IQRNMV+P/Pre=2.50) and the P group (MP/Pre=+2.53 deg; IQRP/Pre=4.50).

  Friedman's test result [χ²(2, N=87)=7.27; p=0.026]. We noted that the median SSA was initially oriented toward the right in the Pre period (MPre=+3.30 deg; IQRPre=4.10) but tended to be slightly deviated leftwards immediately after the interventions (MPost0=+2.45 deg; IQRPost0=4.40; z=2.25; p=0.024) and then 30 minutes afterwards (MPost30=+2.50 deg; IQRPost30=4.85; z=2.49; p=0.013). However, only the NMV+P intervention contributed significantly to these results; only in this group did the period have a significant effect on the median SSA [χ²(2, N=24)=24.25; p<0.001]. More precisely, the SSA was significantly deviated leftwards immediately after the intervention (MNMV+P/Post0=+1.43 deg; IQRNMV+P/Post0=2.13; z=3.96; p<0.001) and then 30 minutes afterwards (MNMV+P/Post30=+2.00 deg; IQRNMV+P/Post30=2.83; z=3.64; p<0.001), relative to the Pre values (MNMV+P/Pre=+3.63 deg; IQRNMV+P/Pre=2.50). The difference between the median SSA at Post0 and at Post30 did not reach the level of significance (z=1.90; p=0.057). In contrast, the three other interventions did not influence significantly the SSA over the course of the three test periods, as shown by the non-significant results of Friedman's test in the NMV group [χ²(2, N=21)=2.57; p=0.28], the NMV+V group [χ²(2, N=22)=0.71; p=0.70] and the P group [χ²(2, N=20)=0.70; p=0.70]. This differential effect of the type of intervention was corroborated by the Kruskal-Wallis analyses computed on the Post-Pre SSA differences. Immediately after the intervention, the SSA variation differed significantly across the groups [H(3, N=87)=25.16; p<0.001]. SDCF pairwise comparison method revealed that the SSA variations were significantly higher in the NMV+P group (MNMV+P/Post0-Pre=-2.43 deg; IQRNMV+P/Post0-Pre=1.18) than in the NMV group (MNMV/Post0-Pre=+1.25 deg; IQRNMV/Post0-Pre=3.95, p<0.001), in the NMV+V group (MNMV+V/Post0-Pre=-0.25 deg; IQRNMV+V/Post0-Pre=2.35, p<0.001) or in the P group (MP/Post0-Pre=-0.80 deg; IQRP/Post0-Pre=2.93, p=0.016). Other comparisons between groups were not significant (ps>0.05). Thirty minutes afterwards, the group effect on SSA variations was also significant [H(3, N=87)=8.02; p=0.046]. The median SSA's Post30-Pre changes were larger (in absolute) in the NMV+P group (MNMV+P/Post30-Pre=-1.73 deg; IQRNMV+P/Post30-Pre=2.10) than in the NMV group (MNMV/Post30-Pre=+0.05 deg; IQRNMV/Post30-Pre=2.15), in the NMV+V group (MNMV+V/Post30-Pre=-0.83 deg; IQRNMV+V/Post30-Pre=3.65) or in the P group (MP/Post30-Pre=-0.53 deg; IQRP/Post30-Pre=3.85). However, only NMV group differed significantly from the NMV+P group (p=0.016; p>0.05 for all other pairwise comparisons) concerning this SSA Post30-Pre change.

Figure .

 . Figure. Median ([IQR] and range) visual angle errors (in degree) during the blindfolded subjective straight-ahead (SSA) finger-pointing test before each intervention (Pre), immediately afterwards (Post0), and 30 minutes afterwards (Post30): "neck muscle vibration, blindfolded" (NMV), "neck muscle vibration with vision" (NMV+V), "neck muscle vibration with visual finger-pointing" (NMV+P), and 'visual finger-pointing' (P). ***: p<0.001

Acknowledgments: The authors thank C. Aubert and O. Dossmann (respectively Trainer and Director at the Institut de Formation en Ergothérapie Lorraine et Champagne-Ardenne, Nancy, France), their occupational therapy students, and those at STAPS Nancy of Université de Lorraine for their participation at this study. The authors also thank R. Weinland and E. Bernard for technical assistance and Dr David Fraser (Biotech Communication SARL, Ploudalmézeau, France) for English editing assistance.

Funding: This work was supported by the Région Lorraine and the Université de Lorraine, France [Contrat d'objectifs avec les Etablissements d'Enseignement Supérieur et de Recherche Lorrains -Exercice 2015].


Marie E Vuillemin

Lionel Muniglia

Michel Linder

Sabine Bouguet-Bonnet

Sophie Poinsignon

Raphael Dos

Santos Morais

Blandine Simard

Cédric Paris

Florentin Michaux

Jordane Jasniewski
email: jordane.jasniewski@univ-lorraine.fr

Polymer functionalization through an enzymatic process: intermediate products characterization and their grafting onto gum Arabic

Keywords: gum Arabic, functionalization, phenol grafting, ferulic acid, oxidation product

The modification of gum Arabic with ferulic acid oxidation products was performed in aqueous medium, at 30 °C and pH 7.5, in the presence of Myceliophthora thermophila laccase as biocatalyst. First, this study aimed to investigate the structures of the oxidation products of ferulic acid that could possibly be covalently grafted onto gum Arabic. HPLC analyses revealed that this reaction produced several oxidation products, whose structures were investigated using LC-MS/MS analyses (liquid chromatography-mass spectrometry with mass

Introduction

Gum Arabic (GA) is a sticky exudate of air-solidified sap from the trunks or tree branches of Acacia trees such as Acacia senegal and Acacia seyal, which essentially provides a friable gum [START_REF] Idris | Characterization of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF][START_REF] Mcnamee | Emulsification and Microencapsulation Properties of Gum Arabic[END_REF][START_REF] Nussinovitch | Exudate gums[END_REF]. GA is mainly composed of D-galactose (39 to 42%), L-arabinose (24 to 27%), L-rhamnose (12 to 16%), D-glucuronic acid, 4-O-Methyl-β-D-glucuronic acid (15 and 16%) and a small amount of proteins (1.5 to 2.4%) [START_REF] Idris | Characterization of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF]. The polysaccharides and proteins present in GA can be divided into three main fractions [START_REF] Idris | Characterization of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF][START_REF] Williams | Spectroscopic and molecular comparisons of three fractions from Acacia senegal gum[END_REF]: arabinogalactan-peptide (AG), arabinogalactan protein (AGP) and glycoprotein (GP). These fractions differ in their molecular weight, protein content and chemical composition [START_REF] Mahendran | New Insights into the Structural Characteristics of the Arabinogalactan-Protein (AGP) Fraction of Gum Arabic[END_REF]Randall, enzymatic modification of gum Arabic with ferulic acid oxidation products in the presence of Myceliophthora thermophila laccase as biocatalyst [START_REF] Vuillemin | Physicochemical characterizations of gum Arabic modified with oxidation products of ferulic acid[END_REF]. This study showed that this functionalization changed GA properties and led to an increase of its antioxidant properties and led to the apparition of a glass transition closed to 39 °C. However, the study did not prove undeniably if the oxidation products of ferulic acid were grafted on GA or only adsorbed onto it.

The main issue with this approach is that the laccase-catalyzed oxidation of ferulic acid is currently not well-controlled. It generates highly reactive unstable radicals that can spontaneously reorganize and may lead to the formation of phenolic oligomers. Many structural changes, including oxidative decarboxylation, may occur, thus increasing the number of possible oxidation product structures. Only a few structures have been fully elucidated, most of the others have not been completely identified yet [START_REF] Ralph | Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls[END_REF]. Furthermore, the existing identification of such products rely mostly on hypothesis based on the products molecular weight obtained by liquid chromatography-mass spectrometry (LC-MS) measurements [START_REF] Aljawish | Laccasecatalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: A green procedure for the synthesis of new compounds[END_REF][START_REF] Carunchio | Oxidation of ferulic acid by laccase: identification of the products and inhibitory effects of some dipeptides[END_REF][START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF]. Yet, for one molecular weight, multiple structures could be proposed. Some studies have demonstrated that several structures of the same molecular weight could coexist, based on nuclear magnetic resonance [START_REF] Ralph | Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls[END_REF]. Thus, this study aimed on one hand to determine the chemical structure of the oxidation products obtained from the oxidation of ferulic acid initiated by the laccase of Myceliophthora thermophila. The functionalization of polysaccharides through enzymatic process is a complex matter because it would require an interaction between the enzyme and polysaccharide, which are generally difficult and limited due to the steric clutter associated with the size of the polymer. As a solution, the laccase-mediated functionalization is proposed. In this enzymatic pathway, only the phenolic compound (i.e. ferulic acid) is initially supported by the enzyme to form reactive species (semi-quinones). These radicals are active species which can either condense with each other or react with nucleophilic function, such as amino groups or carboxyl groups present on the polysaccharide. This mechanism has already been widely studied and although the mechanism of the non-enzymatic reaction step is still poorly understood, experimental evidence supports the hypothesis that quinones can undergo different types of reaction with amines to yield either Schiff-bases or Michael-type adducts, as well as oligomer-forming reactions with other quinones (Kumar et al., 2000, Mustafa et al., 2005[START_REF] Aljawish | Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions[END_REF].

The study of the grafted products is complex, which is why the study was focused on the free products that are easier to study. The assumption was made that they would not be different with or without gum Arabic since the intermediate species are formed before reacting with the polysaccharide. However, it could not be excluded that some rearrangements happen afterwards and that they could differ depending on whether the oxidation products condense with each other or react with gum Arabic.

Those products were expected to be grafted either on the carboxyl or on the amine group of GA. This study aimed to prove the grafting of the oxidation products onto the polymer and to locate these chemical species on GA. HPLC, LC-MS/MS and NMR analysis were used to resolve the structure of reactions products coming from the ferulic acid oxidation by the laccase. Then, NMR spectroscopies have been performed to confirm the grafting of the oxidation products on the polysaccharide.

Experimental

Materials

Gum Arabic (GA) Instantgum AA from Acacia senegal was a gift from Nexira (France).

Ferulic acid (purity about 99%) (FA), anhydrous sodium phosphate dibasic (Na2HPO4), anhydrous potassium phosphate monobasic (KH2PO4), trifluoroacetic acid (TFA), deuterated water and deuterated methanol were purchased from Sigma-Aldrich (France). Analytical grade methanol, ethanol, formic acid and acetonitrile were purchased from Carlo Erba (Milwaukee, WI, USA).

The enzyme used for the oxidation was commercialized as "Novozym® 51003" from Novozymes (Bagsvaerdt, Denmark). It is a fungal laccase from Myceliophthora thermophila, a polyphenol oxidase produced by submerged fermentation of a genetically modified Aspergillus oryzae [START_REF] Berka | Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae[END_REF]. The laccase activity was determined by following the apparition of the oxidation product of syringaldazine (the reference substrate for laccase activity determination). The laccase stock activity was 32 038 ± 1 322 LAMU.g -1 (Laccase Myceliophthora Units.g -1 ).

Methods

Functionalization of gum Arabic

The method used to functionalize GA through a ferulic acid oxidation was adapted from previous protocols [START_REF] Aljawish | Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions[END_REF][START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF][START_REF] Vuillemin | Physicochemical characterizations of gum Arabic modified with oxidation products of ferulic acid[END_REF]. 1 g of GA was dispersed in 45 mL of phosphate buffer (50 mM, pH 7.4). The dispersion was stirred at 450 rpm for 1 h at 30 °C. 5 mL of ferulic acid (50 mM) dissolved in methanol was added to the batch. Once the temperature was stable, 13.5 LAMU.mL -1 of Novozym® 51003 was added to trigger the reaction. The batch was then stirred at 30 °C for 50 min. 150 mL of ethanol (96%) stored at -20 °C was then added to stop the reaction and separate the nonfunctionalized GA from the functionalized GA. The obtained mixture containing functionalized gum Arabic (FGA), non-grafted FA oxidation products (OXP) (soluble in the phosphate buffer/ethanol mixture) and non-functionalized GA (insoluble in the phosphate buffer/ethanol mixture) was then centrifuged at 12000 g for 20 min at 20 °C with a Beckmann centrifuge (Beckman Coulter Inc., Villepinte, France). The precipitate contained nonfunctionalized GA and a part of phosphate buffer salts (verified by FTIR measurements, data not shown), whereas the supernatant contained the functionalized GA and the ferulic acid oxidation products not grafted on GA.

The ethanol was then eliminated with a rotary evaporator BuchiR144 (Buchi SARL, Rungis, France) at a boiling point of 40 °C at 175 mbar. Some amount of water from the buffer solution was also evaporated by decreasing the pressure down to 72 mbar.

Preparation of ferulic acid oxidation products

For the preparation of ferulic acid oxidation products the same protocol as for the functionalization of gum Arabic has been performed but without adding gum. Ferulic acid and OXP were soluble in the mixture of methanol and phosphate buffer. However, at the end of the reaction, 150 mL of ethanol (96%) stored at -20 °C was still added to stop the reaction and to precipitate buffer salts. The supernatant contained the OXP.

Purification of the component

For FGA, the residual solution was dialyzed against ultrapure water with a high-grade regenerated membrane (MWCO 10 000 Da from Membrane Filtration Products Inc.) to eliminate excess salts from the buffer and non-grafted FA oxidation products (the oxidation products are soluble in water up to 5% [START_REF] Vuillemin | Physicochemical characterizations of gum Arabic modified with oxidation products of ferulic acid[END_REF] and they have a molar mass lower than 610 Da (Figure 2)). Dialysis was stopped when the conductivity outside the membrane was equal to the one of ultrapure water, i.e. equal to 1.0 ± 0.2 µS.cm -1 . The same method was used to eliminate excess salt from NGA. For the OXP, they were purified using Sephadex PD-10 pre-packed desalting columns (with Sephadex G-25 resin, GE Healthcare, Buckinghamshire, United Kingdom). 25 ml of ultrapure water was used to equilibrate the column, after what 2.5 mL of solution of OXP in ultrapure water was added. After the sample had entered the packed bed completely, 2.5 mL of ultrapure water was added again to the column. The eluate that was collected contained the OXP, while the salts remained in the column. The dialyzed FGA and NGA and the purified OXP were then freeze-dried. The powders obtained were stored in a desiccator, in the dark, at 4 °C until use.

Solubilization of the products

The solutions were prepared by dissolving FGA, NGA or OXP in ultra-pure water. The solutions were stirred at 400 rpm overnight at 4 °C to ensure total solubilization of the products.

Color modification during the oxidation reaction

During the functionalization of GA and the oxidation of ferulic acid by the laccase from Myceliophthora thermophila, a change of the color of the mixtures was observed. The color evolution of the reaction medium was measured with a spectrocolorimeter CR-5-Konica Minolta (Konica Minolta Sensing, Europe B.V.). Prior to each run, the device was calibrated using two standards (white and black). Every 10 min, CIE (International Commission on Illumination) color parameters (L*, a* and b*) were measured by placing 4 mL of the reaction mixture in the spectrocolorimeter tank. L* defines lightness and ranges from 0% (black) to 100% (white). The a* parameter denotes the red/green value and b* the blue/yellow value.

The color difference (ΔE) between two samples was obtained using Eq.1.

∆ = ∆ + ∆ + ∆ ² (Eq. 1)
2.2.6. Monitoring of the ferulic acid consumption during the oxidation reaction (HPLC)

The oxidation of ferulic acid during the enzymatic reaction was monitored by high performance liquid chromatography (HPLC) (Shimadzu LC10). 100 µL of the reaction medium was collected and placed in 2 mL microtubes containing 900 µL of pure methanol to stop the reaction. Prior to analysis, samples were filtered through a 0.2 µm syringe filter and half diluted in ultra-pure water. 5 µL was injected into the HPLC system. The separation was performed on a reversed-phase GRACE Apollo C18 chromatography column (150 x 2.1 mm -5 µm). The elution method consisted of a two-phase gradient at a flow rate of 0.2 mL.min -1 : a phase A (ultra-pure water/methanol/TFA 80:20:0.1) and a phase B (100% methanol). The gradient used consisted in 2 min of 20% of phase B, then the proportion of phase B was raised from 20% up to 90% during 14 min. The proportion of B was maintained at 90% for 5 min and finally at 20% for 10 min. The UV-VIS absorbance was measured between 190 and 900 nm on a multichannel photo-diode-array detector (SPD-M10A VP). The results were recorded and processed by a chromatogram processing software (LC solution). The amount of AF was measured at its maximum absorption (322 nm) after making a standard ferulic acid calibration from 0.1 to 0.5 mM.

Measurement of the approximate Zeta potential

The approximate Zeta potential of FGA and NGA (1.00% w/v) were calculated from measurement of the electrophoretic mobility at 25 °C, at pH 5.5, using a Zetasizer Nano-ZS instrument and DTS1070 disposable zetacell (Malvern Panalytical, United Kingdom) equipped with a He/Ne ion laser (λ= 532 nm). Measurements were collected on a detector at 173°. The instrument determined the Zeta potential with the Smoluchowski equation (Jayme, Dunstan, & Gee, 1999). All measurements were made in triplicate.

2.2.8. Structural characterization of the modified gum Arabic: Nuclear magnetic resonance analyses Native gum Arabic (NGA) and functionalized gum Arabic (FGA) were characterized by NMR. They were solubilized in deuterated water at concentrations of 5.0% and 4.5% w/v, respectively. The solutions were then analyzed by NMR to determine their molecular structure and the proximity of the atoms from each other, by using a combination of 1D and 2D NMR techniques. Experiments were performed on a spectrometer operating at 9.4 Tesla (Bruker Avance III, frequencies of 400 Mhz and 100.6 MHz for 1 H and 13 C, respectively), using a Bruker 5mm BBFO probe. Pulse widths were 14.1 and 10.5 µs for 1 H and 13 C, respectively. All experiments were performed at room temperature. The 1 H, 13 C decoupled from 1 H, 1 H-13 C HSQC, 1 H-13 C HMBC, 1 H-1 H ROESY and 1 H diffusion NMR experiments were run under standard conditions. For HMBC experiment, a 50 ms waiting period was used for the evolution of long-range coupling, and a value of 3.4 ms for the low-pass J filter.

ROESY was run with a spin-lock of 100ms for mixing. And diffusion measurements were done using standard stimulated echoes with bipolar gradient pulses, with ∆ 300ms, δ 2.3ms, and 64 points for linear increment of the gradient between 0.96 and 47.2 G.cm -1 .

2.2.9. Structural characterization of the oxidation products 2.2.8.1.

Separation and purification of oxidation products

Separation and fractionation of the oxidation products (OXP) solubilized in ultrapure water were performed using high-performance semi-preparative liquid chromatography (Gilson Inc., Middleton, USA). Results were processed using the Trilution @ LC software.

The OXP separation was carried out using a reversed-phase column GRACE Apollo C18 ( 250x 22 mm -5 µm). The detection was carried out at 322 nm on a UV/Vis 156 detector. The elution gradient consisted in a phase A (ultra-pure water and formic acid 100: 0.1) and a phase B (acetonitrile and formic acid 100: 0.1) at a flow rate of 10 mL.min -1 . 1 mL of sample was injected into the column at a concentration of 4% w/v. The gradient consisted in 10% of the phase B for 2 min, then 60% for 78 min, 99% for 5 min and finally 10% for 5 min. Fractions were recovered from the column outlet in glass tubes. The solvents were evaporated using a rotary evaporator BUCHI R144 (BUCHI SARL, Rungis, France). The OXP were freeze-dried and then stored at 4 °C in a desiccator until use.

Determination of the molecular mass of the oxidation products by Liquid

Chromatography-Mass Spectrometry (LC-MS/MS)

The mass spectra of the OXP were obtained with a UHPLC-MS system, which comprised a quaternary solvent delivery pump connected to a photodiode array and a LTQ Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an atmospheric pressure ionization interface operating in electrospray mode (ESI).

10 µl of the mixture were separated on a C18 Alltima (150 * 4.6 mm -5µm) column (Grace/Alltech, Darmstadt, Germany). The flow rate was set at 200 µl.min -1 and mobile phases consisted in water modified with formic acid (0.1%) for A and acetonitrile modified with formic acid (0.1%) for B. Phenolic compounds were eluted using a linear gradient from 5% to 47% of B for 60 min.

The spray voltage was +5.0 kV. The temperature of the heated capillary was set to 300 °C. The flow rates of sheath gas, auxiliary gas, and sweep gas were set (in arbitrary units/min) to 40, 10, and 10, respectively. Capillary voltage was +36 V, tube lens was +80 V, split lens was -44 V, and the front lens was -3.25 V. Mass spectrometric parameters were optimized by infusing a standard solution of isopimpinelline (0.1 g.L -1 ) in mobile phase at a flow rate of 5 µl.min -1 . Parent ions were detected between 100 and 1000 m/z and specific MS n fragmentation spectra (MS 2 , MS 3 ) were carried out in order to obtain structural information on OXP. Data were processed using Xcalibur software (version 2.1, Thermoscientific).

2.2.8.3. Nuclear magnetic resonance analyses OXP were characterized by NMR according to the same method described for NGA and FGA. The only difference was the solvent and concentration used which in this case were a deuterated water/deuterated methanol mixture (50: 50) at a concentration of 20 mM. The structure determination was done using 1 H, J-modulated 13 C, 1 H-1 H COSY, 1 H-13 C HSQC, mm ID x 300 mm, void volume ~6 mL, total volume ~ 12.5 mL, Malvern Panalytical) were mounted in series and equipped with a post-column nylon filter (0.22 µm). The columns were equilibrated with ammonium acetate buffer 350 mM, pH 5.5. The flow rate was 0.35 mL.min - 1 and the temperature was 30 °C. Data were processed with the Omnisec software (v5.12, Malvern Panalytical). The calibration procedure was done with β-lactoglobulin (Sigma) and cross-validations were performed with PEO24k and Dextran standards (Viscotek PolyCal standards, Malvern Panalytical). The refractometer was used as the concentration detector and the refractive index increment value (dn/dc) used to determine the molecular weight was 0.136 mL/g [START_REF] Grein | Structural characterization and emulsifying properties of polysaccharides of Acacia mearnsii de Wild gum[END_REF]. Free-dried dialyzed samples were solubilized in the aforementioned buffer at 10 g.L -1 and filtered through a 0.22 µm PES-filter just before injection.

Results and discussion

Color modification during the reaction

The first event that reflected a significant change during the oxidation of ferulic acid (FA) was a color change from colorless to dark red (pictures from Figure S1). Indeed, it was already shown in a previous study [START_REF] Aljawish | Laccasecatalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: A green procedure for the synthesis of new compounds[END_REF] that the oxidation products of ferulic acid (OXP) were generally colored. Other polymers grafted with ferulic acid oxidation products were gradually modified towards yellow-orange during functionalization [START_REF] Aljawish | Laccasecatalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: A green procedure for the synthesis of new compounds[END_REF][START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF]. On Figure S1, the color difference of the reaction medium during the oxidation reaction of ferulic acid at different time are presented with and without GA. At this stage, it was important to evidence if the presence of GA modify the oxidation process. FGA and OXP color difference (∆E, between t0, the beginning of the reaction and a chosen time t) increased upon time, meaning that changes occurred from the beginning of the reaction until its end (50 min). The values of color difference of FGA were significantly different when compared with those of OXP. The color change was more pronounced without GA (OXP). The 2 hypotheses formulated to explain this difference in coloring would be, on one hand, the presence of GA and, on the other hand, a slightly lower level of oligomerization in the presence of GA which blocks certain reaction intermediates by grafting. For the first hypothesis, GA was not soluble in the buffer (heterogeneous catalysis), so the presence of the polymer could have made the mixture a little turbid, thus changing the color parameters.

However, the tendencies were close, which could indicate that the oxidation products were most probably the same with or without GA.

Monitoring of the ferulic acid consumption during the reaction

In order to verify if GA did not change the laccase activity or the ferulic acid consumption rate, the consumption of ferulic acid in the reaction mixtures was monitored by HPLC throughout the oxidation process. The consumption of ferulic acid during the reaction with or without GA was monitored by assaying its disappearance from HPLC chromatograms at 322 nm (Figure 1). A control was made with GA and ferulic acid without laccase to ensure the FA concentration would not decrease without laccase, i.e. that FA did not adsorb onto or react with GA. The results presented on Figure 1.A indeed confirmed that FA concentration did not decrease upon time without the enzyme. Physicochemical interactions between phenolic acids and polysaccharides have already been observed by other authors [START_REF] Padayachee | Binding of polyphenols to plant cell wall analogues -Part 1: Anthocyanins[END_REF], but in this case, the assumption that ferulic acid would adsorb onto GA chains was ruled out.

However, when laccase was added, the FA concentration decreased rapidly and similarly with or without GA (0.52 ± 0.01 mM FA.min -1 for OXP and 0.52 ± 0.02 mM AF.min -1 for FGA).

These results suggested that the presence of GA did not interfere with the laccase activity and did not affect the reaction rate. Some authors have reported the rate of reaction of phenol substrate would be slowed down by adding a polymer [START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF][START_REF] Sun | Tyrosinase Reaction/Chitosan Adsorption for Removing Phenols from Wastewater[END_REF][START_REF] Wada | Removal of phenols from wastewater by soluble and immobilized tyrosinase[END_REF] but another study on polysaccharide functionalization [START_REF] Aljawish | Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions[END_REF], also performed under heterogeneous catalysis, demonstrated that the presence of the polymer would not change the reaction rate.

On Figure 1A it can be observed that almost all ferulic acid was consummated after 20 min (0.005 ± 0.001 mM remained for OXP and 0.002 ± 0.001 mM for FGA). This result was quite surprising because the color of the reaction still evolved between 20 and 50 min for both reactions (Figure S1). This could be explained by the reaction pathway. Laccase oxidized phenols in ortho or para position into quinones and semi-quinones [START_REF] Witayakran | Synthetic Applications of Laccase in Green Chemistry[END_REF]) that were unstable and could then react with other molecules. Once they are formed, according to the resulting structure, the oxidation products can then react with another ferulic acid molecules or other oxidation products or be oxidized once again by the enzyme.

Numerous structural changes may also occur, for example by oxidative decarboxylation, thus increasing the number of possible structures grafted onto the polysaccharide [START_REF] Aljawish | Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions[END_REF][START_REF] Karaki | Physicochemical characterization of pectin grafted with exogenous phenols[END_REF]. In addition, these molecular reorganizations are often accompanied by an increase in molecular mass leading to the appearance of more sustained colors, which is observed by colorimetry. In order to understand the changes that occurred after FA was consummated, and to follow the number of oxidation products formed and their modification upon time, the absorbance at 322 nm of the oxidation products of ferulic acid upon retention time, all along the reaction, was studied by HPLC. After the peak recorded at 11.4 min associated to ferulic acid (peak area used to follow its disappearance upon time) several other peaks appeared and may then sometimes disappeared related to the apparition or the disappearance of several oxidation products.

3.3.Study of the oxidation products

The absorbance at 322 nm of the mixtures (Figure 1.B) revealed the presence of several peaks that corresponded to different oxidation products. Their retention times were higher than that of ferulic acid (11.4 min), meaning they were more hydrophobic related to the eluent composition. Some products were formed during the reaction but disappeared before the end (for example the peak at a retention time of 14.7 min). At the end of the reaction, a massive group of peaks was observed in the range of retention time from 17.0 to 21.1 min. The oxidation products did indeed change between 20 and 50 min of reaction, thus explaining why the color of the samples would change (Figure S1) even after all FA was consummated.

Each observed absorbance peaks corresponded to one oxidation product which might react with the polysaccharide when GA was present in the reaction media. Their structures were thus further investigated by LC-MS/MS.

Thanks to UV screening at 322nm recorded during more than 60 minutes, 11 oxidation products were readily identified by LC-MS/MS analysis 50 minutes after the enzyme addition as shown in Figure 2 giving their retention time and their exact molecular mass. The peak n°1 corresponded to dehydrated ferulic acid (shown by its molecular mass (177.06 g.mol -1 ) and retention time (26 s)). The peak n°6 has a molecular mass close to the one of ferulic acid (MFA=194.18 g.mol -1 ) so it would seem, therefore, that it corresponded to a molecule with only one phenolic ring with a structure close to the one of ferulic acid. From their molecular weights, OXP n° 2 and 9 were most probably ferulic acid derivatives trimers formed with three derivatives of ferulic acid whereas the majority of the 7 other OXP were obviously ferulic acid derivatives dimers as already observed in previous studies [START_REF] Aljawish | Laccasecatalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: A green procedure for the synthesis of new compounds[END_REF][START_REF] Carunchio | Oxidation of ferulic acid by laccase: identification of the products and inhibitory effects of some dipeptides[END_REF].

Figure 2: Absorbance at 322 nm and retention time of the oxidation products of ferulic acid (at the end of the reaction) and corresponding molecular exact mass (precise until 10 -4 g.mol - 1 ).

In order to investigate further their structure, the OXP were fractionated using highperformance semi-preparative liquid chromatography. Based on the purity and quantity of the OXP obtained for each peak, further LC-MS/MS and NMR investigations were performed only on OXP n° 3, 4, 5, 8 and 9.

The chosen OXP were analyzed by LC-MS/MS. From the exact molecular mass, formula predictions were generated. The most probable formula prediction (the one for which the exact mass was closest to the one measured) was presented in Table 1, along with neutral losses identified in MS 2 and MS 3 spectra. These OXP were also analyzed by NMR ( 1 H and 13 C measurements). Due to problems of concentration and thus NMR sensitivity issues, it was not possible to fully elucidate the structure of all studied OXP. Information obtained from standard NMR measurements ( 1 H and 13 C measurements) are presented in supplementary material (Table S1). This preliminary study revealed four of these five OXP were ferulic acid derivatives dimers which probably kept their aromatic rings along with their methoxy groups. The fragmentation of the OXP n° 9 revealed it was probably a trimer composed of a dimer similar to OXP n° 4 and another ferulic acid derivative. Another interesting result was the OXP fragmentations were different, even for the ones with the same molecular mass (n° 3 and 5). These OXP had different retention times (Figure 2), meaning their hydrophobia was different. Though the most probable formulas for these products were the same, it would be difficult to presume to design their structure solely based on their molecular mass, however this approach is often available in literature.

From these results, it was admitted that most oxidation products were ferulic acid derivatives dimers with two aromatic rings and methoxy groups onto it, meaning the most of the oxidation product mechanism would involve forming ester bound between ferulic acids carboxyl groups and hydroxyl groups (Table S1). Further NMR experiments were conducted on the OXP n° 3 to clarify its structure.

Determination of the structure of the oxidation product n° 3

Since it was the one with the highest signal/noise ratio in 1 H and 13 C measurements, the structure of the OXP 3 was investigated further. 1), which meant that two carbons were probably equivalent. 1 H spectrum of the OXP n°3 was presented on Figure 3.B and 1 H-1 H COSY experiment is presented in supplementary materials (Figure S2). The characteristic signals for methoxy groups were displayed at 3.88 and 3.82 ppm. The compound spectrum also showed six aromatic proton at 6.91 (d; J=8 Hz), 6.84 (d; J=8 Hz), 7.17 (dd; J=8 and 1.7 Hz), 6.77 (dd; J=8 and 1.5 Hz), 7.31 (d; J=1.7 Hz) and 6.90 (d; J=1.5 Hz), characteristics for the H-4, H-15, H-5, H-14, H-7 and H-18 of aromatic part of the compound. All chemical shifts and J-couplings are presented in supplementary materials (Table S2) These results, along with the knowledge of the substrate peaks attributions [START_REF] Sajjadi | Isolation and identification of ferulic acid from aerial parts of Kelussia odoratissima Mozaff[END_REF] led to presume there were two aromatic rings from ferulic acid with the methoxy and the hydroxy groups on them.

Correlations obtained by 1 H-13 C HSQC and 1 H-13 C HMBC measurements confirmed this hypothesis and are presented in supplementary materials (Figure S3). Only one peak was observed in the aldehyde/ketone area of the carbon-13 spectrum (175.7 ppm) leading to the assumption there were C=O equivalent groups. They were both correlated to the proton in C-8. This OXP was then identified to be 2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxy-3methoxyphenyl) methylidene] cyclobutane-1, 3-dione presented on Figure 3, called by the authors cyclobutadiferulone. This molecule possesses a tautomeric form that could potentially be charged in solution (Figure S4), but in NMR measurement only one form was detected. It was not possible to propose a mechanism for this OXP formation, which suggested that it did not result simply from the reaction of two oxidized ferulic acids derivatives. The most probable hypothesis was that one ferulic acid was first oxidized/modified by the enzyme and that this intermediate product would have reacted onto another ferulic acid molecule or a modified one. In order to propose a mechanism for the 2-(4-hydroxy-3-methoxyphenyl)-4-[(4hydroxy-3-methoxyphenyl) methylidene] cyclobutane-1, 3-dione formation, further investigations of the intermediate products that appear and then disappear during the reaction (Figure 1) have to be performed all along the reaction.

However, GA has several sites which may react with the oxidation products previously studied. The OXP could react with these functional groups to be covalently linked to the polysaccharide. The grafting of these products onto GA was thus investigated.

Modification of gum Arabic

The impact of OXP grafting on the molecular weight and hydrodynamic radius of GA has been determined using Size exclusion chromatography (SEC) coupled to a multi-detectors system (UV, LS, IV and RI signals were recorded). NGA was analyzed (Figure 4A) and three peaks were observed, corresponding to the expected GP, AGP and AG fractions [START_REF] Picton | Analysis of a complex polysaccharide (gum arabic) by multi-angle laser light scattering coupled on-line to size exclusion chromatography and flow field flow fractionation[END_REF]. The proportion of each fraction was determined using RI detector (0.7%, 10.6% and 88.7%, respectively). The determination of Mw using light scattering gave Mw of ~3.88 x 10 6 g.mol -1 , ~1.25 x 10 6 g.mol -1 and ~3.33 x 10 5 g.mol -1 for the respective fractions. Finally, the determination of the hydrodynamic radius (Rh) was performed thanks to IV detector, giving values of ~28.2, 18.4 and 8.4 nm (see Table S1 in supporting information for a summary of the derived parameters). It is worth notifying that since no significant UV signal at 280 nm was observed, the determination or estimation of the protein content of each fraction was not possible. Nevertheless, all aforementioned results are in line with the literature [START_REF] Randall | Fractionation and characterization of gum from Acacia senegal[END_REF][START_REF] Sanchez | The Acacia Gum Arabinogalactan Fraction Is a Thin Oblate Ellipsoid: A New Model Based on Small-Angle Neutron Scattering and Ab Initio Calculation[END_REF].

FGA was analyzed in the same way (Figure 4B). The first observation was the disappearance of the peaks corresponding presumably to the GP and AGP fractions. Indeed, only one peak having a retention volume really close to the one of the AG fractions in NGA was observed. It was concluded that the two fractions were eliminated during ethanol precipitation realized after the enzymatic reaction leaving only a purified and functionalized AG fraction. Mw obtained for NGA was ~2.13 x 10 5 g.mol -1 and Rh was 6.6 nm (see Table S3 for a summary of the derived parameters). Such values indicated that a large decrease of Mw of the AG fraction occurred during the purification process and/or that a sub-fraction of AG was purified that was previously observed by HIC purification [START_REF] Atgié | Gum Arabic in solution: Composition and multi-scale structures[END_REF].

Surprisingly, no UV signal at 322 nm was observed, certainly due to the small amount of OXP present on FGA. It was also observed that after filtration through 0.22 PES filter just before injection, the sample was colorless and no longer orange. Interestingly, the nylon postcolumn filter was yellowish after unmounting meaning again that excess of OXP. Excess of unbound OXP could have been stuck onto the filters. Nevertheless, thanks to the purification procedure of FGA, a purified and likely functionalized fraction was isolated, having a remarkably small polydispersity index of ~1.04 (Table S3). It was seen previously that the studied OXP could be charged in solution. Several authors already studied the ferulic acid oxidation products molecular mass and suggested that they would be mostly negatively charged dimers of ferulic acid [START_REF] Adelakun | Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity[END_REF][START_REF] Aljawish | Laccasecatalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: A green procedure for the synthesis of new compounds[END_REF]. If they were grafted onto GA, it should change its charge. ) and more uronic acid groups, which is responsible for its negative charge. This should lead to FGA charge being lower than NGA charge in solution [START_REF] Grein-Iankovski | A comprehensive study of the relation between structural and physical chemical properties of acacia gums[END_REF].

The Approximate Zeta potentials of NGA and FGA in ultrapure water were measured at pH 5.5, 25 °C, at 1 % w/v. At this pH, the uronic groups of NGA and FGA should be deprotonated (pKa of 3.6, [START_REF] Aberkane | Thermodynamic characterization of acacia gum-beta-lactoglobulin complex coacervation[END_REF]) as well as grafted ferulic acid oxidation products (pKa aroud 4.6 [START_REF] Dempsey | Ionisation constants of organic acids in aqueous solution[END_REF]) and the amine groups on NGA should be protonated (pKa2 around 6.5 [START_REF] Grein-Iankovski | A comprehensive study of the relation between structural and physical chemical properties of acacia gums[END_REF]).

The NGA Approximate Zeta potential was -25.8 + 0.6 mV, which was related to the presence of the carboxylic groups of the glucuronic acid. This value decreased to -30.1 + 0.9 mV when GA was functionalized, as it was expected from the absence of the GP and AGP fractions in FGA.

The decrease of the charge (or the increase of the negative charge) of GA after functionalization was the first hint that the oxidation products were grafted onto it, however it was not possible to make hypothesis on where it was grafted. They could possibly be grafted onto amino groups of the proteinaceous fraction by forming a Schiff base bound as for chitosan [START_REF] Aljawish | Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions[END_REF] or onto carboxyl groups of the glucuronic acids by forming an ester as for pectin [START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF]. The negative charge of GA was expected to be stable or to increase after functionalization, whether they replace a carboxylic charge or an amine charge.

Zeta Potential measurements and SEC-MALS analysis evidenced a strong modification of GA by the enzymatic functionalization process attributed to the grafting of ferulic acid derivatives.

Therefore, Nuclear Magnetic Resonance (NMR) was used in order to get better insight of the 508 location of the grafting onto GA. 509 tendency as the ones obtained from SEC-multi-detectors experiments (9.6 nm for NGA and 6.6 nm for FGA). In both cases, the polymer size decreased after functionalization. The different values of hydrodynamic radius may be the result of the experiment being performed in different solvents. Indeed, the Rh can vary in a given solvent because of its ability to increase and expand the polysaccharide coils [START_REF] Antoniou | Solvent effects on polysaccharide conformation[END_REF].

Furthermore, in the area corresponding to OXP signals (6.0 to 7.5 ppm) on the FGA spectrum, two diffusion coefficients were obtained, as indicated by the slope break on the inset of Figure 5.B (filled symbols). These two coefficients indicated that there were two types of OXP. One of the diffusion coefficients was the same as the one of the gum signal of FGA (2 x 10 -11 m 2 .s - 1 , parallel lines between gum and OXP signals from 1000 to 2500 G 2 .cm -2 in the diffusion experiment) and could therefore be attributed to a fraction of OXP linked to the gum. The other coefficient was faster (around 8 x 10 -11 m 2 .s -1 ) and could be attributed to a fraction of OXP that were not directly linked to the functionalized GA. This fraction could correspond to a small fraction of non-grafted oxidation products that were not eliminated during dialysis because they were strongly adsorbed on FGA. Diffusion measurements could thus show that a fraction of OXP was grafted onto FGA, 2D NMR approaches were then performed in order to locate were they were grafted.

HMBC spectra were obtained on the three samples. These spectra were first used in order to obtain information on the carboxyl zone before and after functionalization. Indeed, a direct 13 C measurement was not conceivable because of a lack of sensitivity, indirectly detected 13 C spectra (using 1 H polarization) of the three samples were thus obtained from projections of the HMBC (Figure 6). It appeared on these spectra the additional peaks observed on the functionalized gum corresponded to phenolic groups signals. In a second step, and in order to verify if there was a correlation between the FGA carbons and the protons from OXP, HMBC correlation were finely analyzed (supplementary material, Figure S5). The weak proportions of OXP did not make it possible to visualize a direct correlation between the aromatic 1 H of OXP and a carbon of FGA. Even if only intra-OXP or intra-gum correlations could be observed in the HMBC, carboxyl carbon peak moved from 175.4 ppm in the OXP mixture to 168.2 ppm in the FGA sample, which may indicate that OXP were possibly esterified. ROESY experiments of FGA were recorded (Figure 7). They allowed the visualization of a 1 H-1 H correlation by dipolar effect and thus drove to identify protons that were close in space.

A 1 H-1 H dipolar interaction was visible between the aromatic 1 H of OXP and GA but it was difficult to clearly identify the corresponding 1 H due to the width of the correlation spots and their low intensity. However, a correlation was brought out between one aromatic 1 H of OXP and possibly the methylic protons of FGA galactose units (in C6) or the 1 H in C5 of the glucuronic acid units of GA, meaning the OXP would be grafted either onto them or onto the near carbons. The results obtained in a previous study on pectin modification by grafting OXP of ferulic acid [START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF] with the same pathway also concluded on a grafting onto the C6 carbon of the galacturonate units of pectin. Figure 7: (A) ROESY 1 H-1 H and (B) HSQC 1 H-13 C of functionalized gum Arabic FGA. As a reference for OXP, the signals corresponding to ferulic acid are indicated according to the numbering of the figure in the inset of (B). Intra-OXP correlations are indicated by straight lines. Gum Arabic-OXP correlation is indicated by a dotted line.

To summarize, the pulsed field gradient (PFG) measurements indicated that after dialyses there were still two populations of OXP in solution. One of them could be referred to as "free OXP" and the other one was strongly bound to FGA. Nevertheless, the presence of a chemical bonding between OXP and FGA was not obvious by experimental 2D NMR, due to the complexity of the spectra and especially sensitivity problems because of the low grafting rate of OXP. However, there was a set of clues proving that the grafting was conclusive, the strongest one being that the diffusion coefficient of GA was modified after functionalization and this coefficient was also measured in the spectral area corresponding to the OXP species.

Although, no direct interaction between OXP and GA could be evidenced by HMBC. ROESY correlations proved the proximity of both compounds and made it possible to estimate a location for the site of the grafting: close to the C6 of galactose units or close to the C5 of the glucuronic acid units of GA, which was consistent with what was observed on other polymers [START_REF] Karaki | Functionalization of pectin with laccase-mediated oxidation products of ferulic acid[END_REF].

Conclusions

This study focused on the modification of gum Arabic by an enzymatic pathway. The first result was that GA did not alter the enzymatic oxidation of ferulic compounds since neither the color nor the ferulic acid consumption were modify in the presence of the polysaccharide. Furthermore, it was observed by HPLC that several oxidation products were formed which may then react with GA. The exact mass of each OXP was determined by LC-MS/MS, leading to the conclusion that the majority of the products obtained were ferulic acid dimers which kept their aromatic rings and the O-CH3 groups onto them. Assumptions were made about their respective raw formulas and one of them was clearly identified from NMR experiments as cyclobutadiferulone (2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxy-3methoxyphenyl) methylidene] cyclobutane-1,3-dione). Further experiments will have to be carried out in order to make it possible for all OXP structures to be elucidated and to propose a mechanism for their formation. However, the grafting of oxidation products onto GA modified its charge (its approximate Zeta potential decreased) and only one fraction was recovered after functionalization instead of the three of the native gum. SEC-MALS analysis indeed evidenced that only AG fraction remained but with lower molecular weight and polydispersity. From NMR analysis, oxidation products were undeniably present on modified GA, probably close to the glucuronic acid C5 carbon or close to the galactose C6 carbon. In spite of the complexity of such a system due to the number and variability of the ferulic acid oxidation products, the complexity of GA with its three fractions and its high polydispersity, the combination of various well-chosen technics allowed the confirmation of the grafting of ferulic acid derivative onto GA and a better knowledge on the laccase-assisted modification of polysaccharide.

Figure 1 :

 1 Figure 1: (A) Ferulic acid (FA) concentration in phosphate buffer (50 mM, pH 7.4, 30 °C) containing ferulic acid + gum Arabic + laccase (△), ferulic acid + laccase without gum Arabic (•) and ferulic acid + gum Arabic without laccase (control) (■) and (B) Chromatogram at 322 nm of the oxidation products of ferulic acid at different reaction times, from the beginning of the reaction to the end of the reaction (50 min).

Figure 3 :

 3 Figure3: 13 C (A) and 1 H (B) spectra of the OXP n°3 (cyclobutadiferulone) (9.4T, ambient temperature, solvent D2O/deuterated methanol) with the peaks' attribution (the chemical shift, J-coupling and 1 H-1 H COSY and 1 H-13 C HSQC spectra of the OXP n°3 are presented in supplementary material)

Figure 4 :

 4 Figure 4: Size exclusion chromatography characterization (refractive index in filled line, Mw Δ, Rh □) of native gum Arabic (NGA) (A) and functionalized gum Arabic (FGA) (B). AG = arabinogalactan-peptide, AGP = arabinogalactan protein and GP = glycoprotein.

Furthermore

  , the FGA being a functionalized AG fraction, it should contain less amine groups

Figure 5 :

 5 Figure 5: (A) 1 H NMR spectra of oxidation products (OXP), functionalized gum Arabic (FGA) and native gum Arabic (NGA) and (B) 1 H NMR spectra of functionalized gum Arabic (FGA). In the inset: evolution of the signal (logarithmic scale, as a function of the gradient g applied) obtained by pulsed field gradient measurement, for two different areas of the spectra. (•) Signal of OXP from 6.0 to 7.5 ppm; (○) signal of gum Arabic from 5.0 to 5.5 ppm. The diffusion coefficients were obtained from these evolutions.

Figure 6 :

 6 Figure 6: 13 C NMR spectra obtained from the projections of HMBC recorded on oxidation products (OXP), native gum Arabic (NGA) and functionalized gum Arabic (FGA).

Table 1 :

 1 Peak number from Figure2, neutral losses from MS 2 and MS 3 spectra, most probable formula prediction.

	Peak number	Exact mass	Most probable formula prediction	Neutral loss (MS 2 ) Neutral loss (MS 3 )
	3	340.0945	C 19 H 16 O 6	CO 2		CH 3 OH
	4	386.1000	C 20 H 18 O 8	CO 2		H 2 O
	5	340.0945	C 19 H 16 O 6	CO		H 2 O
	8	342.1101	C 19 H 17 O 6	2H 2 O		None
	9	588.1608	C 32 H 28 O 11	M=386 g.mol -1 OXP 4	=	None

H-13 C HSQC-TOCSY, and 1 H-13 C HMBC.

2.2.8.4.Size-exclusion chromatography coupled to multi-detectors SEC experiments were performed with a HPLC pump (LC10AD, Shimadzu) coupled to an autosampler (Autosampler VE 2001, Malvern Panalytical) and a multi-detectors system recording UV, light scattering (RALS/LALS,) intrinsic viscosity and refractive index signals(Viscotek TDA305, Malvern Panalytical). Two SEC columns (A4000-A6000, 10 or 13 μm, 8

We would like to thank Nexira for kindly providing the Gum Arabic used in this article.

We also thank Aurélie Seiler for the technical support and the Plateforme de RMN de l'Institut Jean Barriol, Université de Lorraine. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

1 H NMR experiments were performed on three samples: NGA, FGA and OXP (Figure 5.A).

The signals of NGA and FGA were crowded between 3.0 and 6.0 ppm, which is typical of polysaccharides and reflected the presence of the sugar residues [START_REF] Nie | The core carbohydrate structure of Acacia seyal var. seyal (Gum arabic)[END_REF]. Those signals have not been deteriorated after functionalization, meaning that the polymer main structure was preserved. On the OXP spectrum, the peaks between 6.0 and 7.5 ppm were characteristic of aromatic proton [START_REF] Sajjadi | Isolation and identification of ferulic acid from aerial parts of Kelussia odoratissima Mozaff[END_REF]. On the spectrum of dialyzed FGA, broad signals were visible in this aromatic compound area (6.0 to 7.5 ppm), which corresponded to the grafted OXP. The signal was not very intense due to the weak grafting rate and to the broadening due to the loss of mobility occurring in the grafting.

Pulsed field gradient (PFG) is a common NMR technique to study the diffusion of polymers [START_REF] Callaghan | Influence of polydispersity on polymer selfdiffusion measurements by pulsed field gradient nuclear magnetic resonance[END_REF][START_REF] Dixon | Modified Pulsed-Field Gradient NMR Experiments for Improved Selectivity in the Measurement of Diffusion Coefficients in Complex Mixtures: Application to the Analysis of the Suwannee River Fulvic Acid[END_REF][START_REF] Oostwal | Chain self-diffusion in aqueous salt-free solutions of sodium poly(styrenesulfonate)[END_REF]. Such measurement was made on the NGA and FGA samples (Figure 5.B).

NGA was found to have a unique diffusion coefficient (around 1 x 10 -11 m 2 .s -1 , data not shown). Results obtained on the FGA sample (Figure 5.B) were analyzed considering two distinct areas of the 1 H one-dimensional NMR spectrum. In the area corresponding to the gum signal only (between 5.0 and 5.5 ppm), a unique diffusion coefficient was obtained, as shown by signal monotonous evolution obtained in the inset of Figure 5.B (open symbols). The obtained value of 2 x 10 -11 m 2 .s -1 , slightly higher than the one obtained with NGA, indicated a decrease of the polymer size and/or a decrease in medium viscosity after functionalization.

The evolution of the hydrodynamic radius determined from the diffusion coefficient using the Stockes-Einstein equation [START_REF] Gaylord | Physical chemistry of macromolecules[END_REF] are in good correlation with the ones obtained from SEC-multi-detectors experiments (Table S1). Indeed, the calculated Rh based on the diffusion coefficient (21.8 nm for NGA and 10.9 nm for FGA) showed the same

AUTHOR INFORMATION

Corresponding Author Email: jordane.jasniewski@univ-lorraine.fr

ACKNOWLEDGMENTS AND FUNDING

The authors acknowledge support of the LIBio by the "Impact Biomolecules" project of the "Lorraine Université d'Excellence" (Investissements d'avenir -ANR).