Hassana Hsein

Ghislain Garrait

Muhammad Ashraful Mumin

Eric Beyssac
email: eric.beyssac@udamail.fr

Valérie Hoffart

Atomization of denatured whey proteins as a novel and simple way to improve oral drug delivery system properties

In the sphere of drug delivery, denatured whey protein (DWP) has in recent times gained press. However, to date, no scalable and affordable dosage form has been developed. The objective of our study was to evaluate the potential use of spray-dried DWP as a ready to use excipient for oral drug delivery. Therefore, solid state, FTIR spectra and wettability were studied. Dissolution, mucoadhesion and the effect on paracellular permeability were also evaluated. The spray-dried DWP particles were spherical with 4 µm mean diameter. Further, relative to native WP, the spray-dried DWP particles bore reduced wettability, and their structure was characterized by the exposure of a high amount of free thiol and by the formation of intermolecular β-sheets. The DWP powders were mucoadhesive, enzymatic inhibitors, biocompatible and they induced the opening of tight junctions. Our study shows great potential for the use of spray-drying as a technique to modify the dissolution rate of drugs and enhance the oral bioavailability of molecules. That is, the use of spray drying as a single step ready to use DWP excipient

Abbreviations

DMEM medium Dulbecco's modified eagle medium DWP denatured whey protein DWP-sp spray-dried denatured whey protein particles EE Encapsulation efficiency NWP native whey protein NWP-sp spray-dried native whey protein particles PEG polyethylene glycol WP whey protein Keywords Whey protein, denaturation, spray-drying.

. Even in light of these advantages, very few studies have explored spray-drying of DWP and those which did were not conducted with pharmaceutical applications in mind. For example: Picot and Lacroix increased survival of Bifidobacterium [6]; Koga et al. assessed stabilization of trans-resveratrol [17]; Wang et al. studied stabilization of DHA fish oil [18]; Liu et al. evaluated solubility enhancement of curcuma [19]; and Esfanjani et al. succeeded in encapsulating saffron extracts into DWP/pectin walls [20]. So, many applications for nutraceutical uses but little is known about the biopharmaceutical properties of spray-dried DWP (DWP-sp) and their benefits in oral drug delivery.

For the first time, we propose a new direction for the use of DWP. We propose the spraydrying of DWP for drug encapsulation and oral delivery applications. We do so with the goal of improving oral drug bioavailability.

In testing our hypothesis as to the efficacy of the spray drying methodology, numerous characterizations were performed on the obtained WP-sp powder. These characterizations sought to determine the impact of spray-drying on the DWP structure/morphology and physico-chemical properties such as crystallinity, free thiol content, solubility and wettability. Also studied, with specific regard to oral administration and biopharmaceutical considerations, were apparent dissolution, mucoadhesivity, enzymatic inhibitory effect and the effect on Caco-2 cell permeability. Finally, a model drug, BCS (Biopharmaceutical Classification System) class I, was co-sprayed with DWP to evaluate the impact of encapsulation into DWP, by spray-drying, on the drug release profile.

Introduction

Whey protein (WP) is a valuable milk component, of high nutritional value, that has long been used in the food industry, serving multiple functions including emulsifying, gelling and foaming. In recent times, the nutraceutical and pharmaceutical fields have shown greater interest in WP owing to the encapsulation and protective properties of bioactive molecules therein found. The potential of WP as a nutraceutical delivery system was initially explored with regard to vitamins such as A, D, E, riboflavin [START_REF] Beaulieu | Elaboration and characterization of whey protein beads by an emulsification/cold gelation process: application for the protection of retinol[END_REF][START_REF] Diarrassouba | Effects of gastrointestinal pH conditions on the stability of the β-lactoglobulin/vitamin D3 complex and on the solubility of vitamin D3[END_REF][START_REF] Somchue | Encapsulation of α-tocopherol in protein-based delivery particles[END_REF][START_REF] Chen | Food protein-based materials as nutraceutical delivery systems[END_REF] and minerals such as iron [START_REF] Remondetto | Iron availability from whey protein hydrogels: an in vitro study[END_REF]. Probiotics were also successfully encapsulated into WP microparticles, which contributed to increase their survival in the gastro-intestinal tract [START_REF] Picot | Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt[END_REF]. Indeed, encapsulation in such matrix system resulted in specific protection from acidic pH, enzymatic degradation, and oxidation to ensure bioactivity [START_REF] Chen | Food protein-based materials as nutraceutical delivery systems[END_REF]. WP then gained interest in the sphere of drug delivery, and controlled release. This was due to WP's ability to selectively release molecules such as ketoprofen [START_REF] Betz | Preparation of novel whey protein-based aerogels as drug carriers for life science applications[END_REF], theophylline [START_REF] Hébrard | Investigation of coated whey protein/alginate beads as sustained release dosage form in simulated gastrointestinal environment[END_REF] or insulin [START_REF] Déat-Lainé | Efficacy of mucoadhesive hydrogel microparticles of whey protein and alginate for oral insulin delivery[END_REF], in response to environmental stimuli such as changes in pH [START_REF] Kashyap | Hydrogels for pharmaceutical and biomedical applications[END_REF].

Materials and Methods

Materials

Whey protein isolate (WPI ® 895, protein 93.5%, fat 0.39%, moisture 4.7%, lactose monohydrate 0.2%) was obtained from Fonterra® dairy products, France. Theophylline was purchased from Cooper chemical company, Melun, France. Cell culture products and dissolution buffers components were purchased from Fisher scientific Elancourt, France. All other materials were purchased from Sigma-Aldrich, France.

Preparation of spray-dried WP powders 2.2.1. WP polymeric solutions

NWP solutions (10%, w/w) were prepared by re-hydrating whey protein isolate powder (unrefined WP) in deionized water by gentle magnetic stirring for 1 h at room temperature and then allowed to rest for 2 h to ensure complete hydration of proteins to form a solution. To prepare DWP solutions, NWP solutions were heated at 80°C for 40 min [START_REF] Lefèvre | Molecular differences in the formation and structure of fine-stranded and particulate β-lactoglobulin gels[END_REF]. The solutions were cooled overnight at room temperature. All WP solutions were adjusted at pH 7.0 with NaOH 1M. Viscosity of all polymeric solutions was measured using a Brookfield DVII+ viscometer at 25˚C, using a SC4-18 spindle at 100 rpm (Brookfield Engineering laboratories, Inc., USA).

Preparation of particles by spray-drying

Prepared solutions were spray-dried using a Buchi Mini Spray Dryer (B-290, Buchi® Labortechnik, Flawil, Switzerland), which employs a system of co-current drying via a twofluid nozzle mechanism. The nozzle size was 1.5 mm and the nozzle cleaning procedure was set at 5 throughout each run. Aspiration was maintained at 100%. Spray drying was performed in the following conditions [START_REF] Hsein | Denatured Whey Protein Powder as a New Matrix Excipient: Design and Evaluation of Mucoadhesive Tablets for Sustained Drug Release Applications[END_REF]: inlet temperature (160˚C), pump rate (5.7 mg.min -1 ) and the spray gas flow rate or Q-flow (50 mmHg or 600 L/h). Outlet temperature was noted for every batch. All powders were then stored in desiccator and yield was calculated as following:

Yield (%) =
amount of collected powder (g) amount of initial powder (g) x 100

(1)

Physico-chemical characterization of unloaded spray-dried WP particles 2.3.1. Residual humidity

Moisture content of spray-dried powders was immediately determined once the powder was collected from the dryer by thermogravimetric method using an infrared balance (Mettler Toledo LJ16 moisture analyser, Viroflay, Switzerland). Two grams of powder were weighed and residual humidity was determined after 10 min of heating at 80°C.

Particle size analysis

Particle sizes of powders were determined using a laser light diffraction granulometer (Mastersizer S, Malvern Instruments, Malvern, UK) outfitted with an MS64-dry powder feeder sample unit. The lens used was 100 mm, the active beam length was set to 10 mm and the analysis model used was polydisperse. The results were volume based and the mean volume diameter, D [START_REF] Somchue | Encapsulation of α-tocopherol in protein-based delivery particles[END_REF][START_REF] Diarrassouba | Effects of gastrointestinal pH conditions on the stability of the β-lactoglobulin/vitamin D3 complex and on the solubility of vitamin D3[END_REF], was selected to represent the average diameter of particles.

Scanning electron microscopy (SEM)

The external morphology of particles was studied by scanning electron microscopy (JEOL, JSM-6060LV, Tokyo, Japan). The samples were fixed onto double faced carbon tapes and sputtered with thin film of Palladium-Gold for 60 s using the following device: JEOL JFC-1300. Specimens were examined at 5 KV with different magnifications.

FTIR analysis

Structure of WP was analyzed by infrared spectrometer (IRAffinity-1S FTIR, Shimadzu, Kyoto, Japan) equipped with attenuated total reflectance accessory (ATR-FTIR, Miracle 10). Spectra of unrefined WP and spray-dried samples were collected in absorbance mode from 400-4000 cm -1 , with 200 scans at 4 cm -1 resolution. Deconvolution of spectra was performed in the amide I region (1600-1700 cm -1 ) to study secondary structure of WP using the Happ-Ganzel apodization, 20 cm -1 band width and 0.1 cm -1 pathlength using IR-Solution software.

Free thiol content determination

The amount of free thiol in WP samples was determined spectrophotometrically with Ellman's reagent (Pierce Biotechnology, Rockford, USA) according to the manual instructions guide [START_REF] Hsein | Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength[END_REF][START_REF] Guggi | Systemic peptide delivery via the stomach: in vivo evaluation of an oral dosage form for salmon calcitonin[END_REF]. First, NWP-sp and DWP-sp powders were dissolved in deionized water (pH 6.8) at 20 mg.mL -1 and 250 μL of the obtained WP solutions were mixed with 2500 μL of reaction buffer and 50 μL of Ellman's reagent. The samples were incubated for 15 min at room temperature followed by measurement of absorbance at 412 nm. The number of thiol groups was calculated using a molar extinction coefficient for 5-thio 2-nitrobenzoic acid (TNB) of 14150 M -1 .cm -1 at 412 nm (Ellman's reagent manual instructions guide, Pierce Biotechnology).

Contact angle measurements

Contact angle measurements were done to analyze the wettability of WP powders [START_REF] Grundke | Experimental studies of contact angle hysteresis phenomena on polymer surfaces -Toward the understanding and control of wettability for different applications[END_REF][START_REF]Contact Angle, Wettability and Adhesion[END_REF] using Theta Lite goniometer (Attension, Stockholm, Sweden) equipped with IC capture software. Samples (200 mg of spray-dried powders) were compacted (1.5 t of pressure during 30 s) prior to experiments in order to get a smooth surface allowing accurate measurements. Then, samples were placed on a 3D manipulator plate which was adjusted to be straight and horizontal and a micro-syringe containing deionized water was mounted above the samples. Data was acquired as one droplet (10 µL) was deposited from the microsyringe on the sample. Resulting images were analyzed by ImageJ program to calculate contact angle. Each sample was prepared in triplicate and two independent measurements were made for each surface.

Solubility study

Solubility study was performed according to the excess substrate addition method (Florence and Attwood, 2006; Martin and Sinko, 2011). NWP-sp or DWP-sp were added in excess to 20 mL deionized water or pH 1.2, 4.5, 6.8 or 7.8 european pharmacopeia buffers under agitation. After 24 h, suspensions were centrifuged 10 000 rpm, 10 min and supernatants were analyzed according to BCA method (Bio Basic Inc, Markham Ontario, Canada) to determine WP solubility.

Biopharmaceutical characterization of unloaded spray-dried WP particles for oral administration 2.4.1. Apparent dissolution of WP powders

Dissolution profiles of NWP-sp and DWP-sp powders (300 mg) were investigated using an automated flow-through cell dissolution apparatus (USP 4, Sotax, Aesch, Switzerland) with 200 mL of pH 1.2 or 6.8 USP buffer at 37°C in a closed loop setting (22.6 mm cells). Rate was fixed at 20 mL.min -1 and a whatman GF/F filters were used in each cell. Samples (1 mL replaced by fresh buffer) were withdrawn at set interval times up to 8 h and centrifuged at 5000 rpm during 5 min. Quantities of released WP were calculated using a UV spectrophotometer (Jenway 7315, Staffordshire, United Kingdom) at 280 nm.

Mucoadhesive properties

Mucoadhesion of WP powders was evaluated using the technique of non-everted gut sacs [START_REF] Takeuchi | Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems[END_REF] in accordance with The Code of Ethics of the EU Directive 2010/63/EU for animal experiments. After anesthesia, rat jejunum was cut into equal fragments of 10 cm and washed several times with saline before filling it with 2 mL of suspension containing either 500 mg of NWP-sp or DWP-sp powders in Krebs Henseleit buffer (D-glucose 2 g.L -1 , magnesium sulfate 0.141 g.L -1 , potassium phosphate monobasic 0.16 g.L -1 , potassium chloride 0.35 g.L -1 , sodium chloride 6.9 g.L -1 , calcium chloride dihydrate 0.373 g.L -1 and sodium bicarbonate 2.1 g.L -1 ). Then, gut sacs were sutured and placed in tissue chambers containing 15 mL of warmed (37°C), oxygenated (95% O 2 /5% CO 2 ) Krebs Henseleit modified buffer for 1 h . After this stipulated time, sacs were opened, content collected and then dried in an incubator at 60°C until constant weight to determine the remaining quantity of WP. The rate of adhered powder onto the sac was determined by subtracting the residual mass from the initial mass via Equation [START_REF] Diarrassouba | Effects of gastrointestinal pH conditions on the stability of the β-lactoglobulin/vitamin D3 complex and on the solubility of vitamin D3[END_REF].

Powder mucoadhesion (%) = Residual mass Initial mass x 100 (2)

Enzymatic inhibition properties

Effects of WP-sp powders on enzymatic activity was studied with trypsin and α-chymotrypsin according to Ameye et al procedure [START_REF] Ameye | Optimisation of an in vitro procedure for the determination of the enzymatic inhibition potency of multifunctional polymers[END_REF].

α-chymotrypsin inhibition activity

NWP-sp or DWP-sp were dispersed in Tris HCL pH 7.8 buffer at 1% final concentration (w/w). The substrate N-benzoyl-L-tyrosine p-nitroanilide (BATPNA) was dissolved at 1.18 mM in dimethylformamide (DMF) while α-chymotrypsin from bovine pancreas was dissolved in 10 mM HCl. Then 1 mL of WP solutions, 1 mL of BATPNA and 40 UI of α-chymotrypsin were mixed at 37°C for 30 min before stopping the reaction by the addition of 1% trichloroacetic acid. Finally, the metabolite p-nitroaniline formed was measured spectrophotometrically at 405 nm. Control experiments were conducted using exactly the same protocol but without addition of WP. The activity of α-chymotrypsin in control was taken as 100% and the activity of partially α-chymotrypsin incubated in the presence of WP was expressed relative to 100% activity calculated comparing the absorption of reaction mixtures.

Trypsin inhibition activity

Same WP-sp powders were dispersed in pH 7.4 phosphate buffer at 1% final concentration (w/w). The substrate N-benzoyl dl-arginine p-nitroanilide (BAPNA) was dissolved at 20 mM in dimethylformamide (DMF) while trypsin was dissolved in 10 mM HCl. Then 1 mL of WP solutions, 1 mL of BAPNA and 30 UI of trypsin were mixed at 37°C for 30 min before stopping the reaction by the addition of 1% trichloroacetic acid. Finally, the percentage of inhibition was calculated in an exactly similar manner as applied in the case of α-chymotrypsin inhibition assay.

Cytotoxicity and permeability studies of PEG 4000 on Caco-2 cells

The Caco-2 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Polycarbonate transwell permeable supports (0.4 µm pore size) were seeded with 5.10 5 cell per well at passage 35 and incubated for 21 days with DMEM medium (Dulbecco's modified eagle medium supplemented with 15% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine. Cytotoxicity studies were then performed to evaluate the viability of cells in the presence of NWP-sp and DWP-sp powders at 0.5% (w/v). The viability of the cells was evaluated by trypan blue exclusion (final concentration of 0.2% in PBS) [START_REF] Déat-Lainé | Whey protein and alginate hydrogel microparticles for insulin intestinal absorption: evaluation of permeability enhancement properties on Caco-2 cells[END_REF].

Transport studies were also done after complete cells differentiation and only wells exhibiting transepithelial electrical resistance (TEER, electrode STX2, EVOMX World Precision Instruments, Sarasota, FL, USA) greater than 600 Ω.cm 2 were used. Before experiment, medium was withdrawn and cells monolayers were washed twice with 1 mL PBS buffer.

Then 500 µL of the following samples were added to the apical side of cells monolayers: WPsp powders dispersed at 0.5% (w/v) in HBSS buffer containing 1 mg.mL -1 PEG 4000 as a model molecule [START_REF] Linnankoski | Paracellular porosity and pore size of the human intestinal epithelium in tissue and cell culture models[END_REF][START_REF] Shah | Role of 99mTc-mannitol and 99mTc-PEG in the assessment of paracellular integrity of cell monolayers[END_REF] while basolateral side contained only HBSS buffer. Reference corresponded to untreated cells. Cells were incubated at 37°C during 120 min. At predetermined times, TEER was measured and 500 µL were taken out from basolateral side and replaced by fresh HBSS. Passage of PEG 4000 was determined on withdrawn samples by colorimetric method as described by Nag et al. 1996 [START_REF] Nag | A colorimetric assay for estimation of polyethylene glycol and polyethylene glycolated protein using ammonium ferrothiocyanate[END_REF]. TEER was recorded throughout incubation and during 24 h for recovery period as a measure of cell viability and monolayer integrity. At the end of the experiment, permeability studies were stopped by removing the apical medium and washing the cells three times with PBS. Immediately fresh medium incubated at 37°C was replaced. The monolayers were allowed to regenerate for 24 h at 37°C in an atmosphere of 95% air and 5% CO2 at 90% relative humidity.

Results are expressed as apparent permeability coefficient (Papp) and variation of TEER (%) compared to initial values, calculated according to Equation ( 3) and [START_REF] Chen | Food protein-based materials as nutraceutical delivery systems[END_REF].

TEER (%) = TEER (t) TEER (t0) x 100 (3)
P app (cm. s

-1 ) = ( dQ dt ) × 1 A . C0 (4)
where dQ/dt is the flux of tested product across the monolayer (mol.s -1 ), C 0 is the initial concentration in the apical compartment (mol.mL -1 ), and A is the surface area of the monolayer (1.12 cm 2 ). The Papp values are expressed as cm.s -1 . The Papp values were calculated for the 120 min period of the experiment.

Statistical analysis

All experiments, except SEM, were done in triplicate. Values are presented as mean ± standard deviation (SD) and statistical comparisons were performed using analysis of variance (ANOVA). p < 0.05 was used as the level of significance (p < 0.01 was considered to be very significant).

Results and discussion

Production of DWP particle powder

Spray-drying was used to produce DWP particles intended to be a biopolymer wall and matrix material for drug delivery. Four variables (yield, residual humidity, particle size and outlet temperature) were consequently measured (Table 1). Yield was about 80%, satisfying for the laboratory scale. Residual humidity was under 5%, in accordance with values recommended in the pharmacopeia for solid dosage forms to ensure good storage of product without mold development and bacterial growth [START_REF] Maa | Effect of Spray Drying and Subsequent Processing Conditions on Residual Moisture Content and Physical/Biochemical Stability of Protein Inhalation Powders[END_REF]. Moreover, particle shape and size were monitored, since these can influence drug release kinetics. Laser diffraction showed that mean particles size D [START_REF] Somchue | Encapsulation of α-tocopherol in protein-based delivery particles[END_REF][START_REF] Diarrassouba | Effects of gastrointestinal pH conditions on the stability of the β-lactoglobulin/vitamin D3 complex and on the solubility of vitamin D3[END_REF] was around 4 µm. This result was confirmed by SEM where spherical particles were identified with two populations: under 3 and around 6 µm (Fig. 1). These distributions are characteristic of WP-sp powders and are similar to those reported in literature [START_REF] Koga | Stability of Trans-Resveratrol Encapsulated in a Protein Matrix Produced Using Spray Drying to UV Light Stress and Simulated Gastro-Intestinal Digestion[END_REF][START_REF] Esfanjani | Nano-encapsulation of saffron extract through double-layered multiple emulsions of pectin and whey protein concentrate[END_REF]. Evidence of indentation and wrinkles on powder surface (Fig. 1) might be due to the rapid evaporation and cooling process during drying [START_REF] Rosenberg | A Scanning Electron Microscopy Study of Microencapsulation[END_REF] and to the protein nature of the polymeric matrix [START_REF] Esfanjani | Nano-encapsulation of saffron extract through double-layered multiple emulsions of pectin and whey protein concentrate[END_REF][START_REF] Fäldt | Spray-dried whey protein/lactose/soybean oil emulsions. 2. Redispersability, wettability and particle structure[END_REF]. The same observations were made by Khem et al [START_REF] Khem | The behaviour of whey protein isolate in protecting Lactobacillus plantarum[END_REF]and Shen and Quek [START_REF] Shen | Microencapsulation of astaxanthin with blends of milk protein and fiber by spray drying[END_REF].

Concerning outlet temperature, recorded values (72 ± 1 °C) are acceptable since heating time is very short. Studies showed that even thermolabile proteins such as insulin remained stable and bioactive at these temperatures [START_REF] Maltesen | Quality by design -Spray drying of insulin intended for inhalation[END_REF].

Nevertheless, outlet temperature was higher with NWP and thus the yield (approx. 87%) (Table 1) in link to lower viscosity of NWP compared to DWP solutions (2 vs. 47 cP). In fact, the nozzle plays the role of a heat exchanger between gas flow and feed solution, thereby when viscosity increased, energy supplied to the nozzle should overcome larger viscous forces resulting in decreased outlet temperature and yield [START_REF] Anandharamakrishnan | Effects of Process Variables on the Denaturation of Whey Proteins during Spray Drying[END_REF]. Viscosity appeared clearly as a key parameter to optimize, if necessary, product stability and yield.

It is noteworthy to mention that, despite the theory of aggregate formation following denaturation [START_REF] Donovan | Thermal Denaturation and Aggregation of Whey Proteins[END_REF], in both cases, particle size distributions were similar.

Physico-chemical properties of spray-dried DWP particles

FTIR

FTIR spectra were performed in order to get molecular information about the conformational behavior of WP following denaturation and atomization.

As shown in Fig. 2a, spectra of NWP presented five major absorption peaks in the range of (i) 1200-1350 cm -1 , related to combination of N-H in-plane bending with C-N stretching vibrations (amide III); (ii) 1400-1550 cm -1 , associated to N-H bending (amide II); (iii) 1600-1700 cm -1 , governed by stretching vibration of C=O and C-N groups (amide I); (iv) 2850-2980 cm -1 , assigned to symmetric and asymmetric C-H stretching vibrations; and (v) 3000-3600 cm -1 , attributed to free and bound O-H and N-H groups [START_REF] Ramos | Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefrom[END_REF]. Deconvolution spectra were recorded in the amide I region (1600-1700 cm -1 ) since this region is sensitive and indicative of the protein secondary structure (Fig. 2b) [START_REF] Beaulieu | Elaboration and characterization of whey protein beads by an emulsification/cold gelation process: application for the protection of retinol[END_REF][START_REF] Byler | Examination of the secondary structure of proteins by deconvolved FTIR spectra[END_REF]. The amide I band of unrefined WP contained five individual components; their assignments are well-described in the literature.

The strongest peak at 1631 cm -1 is characteristic of amide groups involved in the extended βsheet structure [START_REF] Allain | Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy[END_REF]. Bands at 1647 and 1666 cm -1 are representative of α-helix/unordered structures and turns respectively. Component at 1678 cm -1 is attributed to β-sheet while that at 1689 cm -1 correspond to intermolecular anti-parallel β-sheet. WP-sp in the chosen operating conditions conserved the secondary structure as seen in Fig.

2b [START_REF] Byler | Examination of the secondary structure of proteins by deconvolved FTIR spectra[END_REF][START_REF] Fang | Conformation of β-Lactoglobulin Studied by FTIR: Effect of pH, Temperature, and Adsorption to the Oil-Water Interface[END_REF][START_REF] Lefèvre | Molecular description of the formation and structure of plasticized globular protein films[END_REF]. However, denaturation induced a dramatic alteration in the amide I band characterized by a featureless broadband between 1630 and 1680 cm -1 indicating the loss of native structures and more precisely the unfolding of polypeptide segments [START_REF] Gilbert | Characterization and evaluation of whey protein-based biofilms as substrates for in vitro cell cultures[END_REF]. Though, the αhelix/unordered (1647 cm -1 ) and turns (1666 cm -1 ) structures were barely conserved. In addition, the strongest band assigned at 1632 cm -1 shifted to 1628 cm -1 which corresponds to the appearance of intermolecular β-sheets resulting from aggregation instead of intramolecular β-sheets [START_REF] Lefèvre | Molecular description of the formation and structure of plasticized globular protein films[END_REF]. This shift occurred simultaneously with an increase in the 1685 cm -1 component showing that additional intermolecular antiparallel β-sheets are formed. Thus, the main secondary conformation of DWP-sp was antiparallel β-sheets characterized by unfolding and aggregation of proteins. This structure was similar to FTIR results obtained in our previous study on DWP powder obtained by polymeric solution freeze-drying [START_REF] Hsein | Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength[END_REF] indicating that heat elements during spray-drying had no influence on DWP-sp structure.

Free thiol determination

The number of free thiol was determined in order to evaluate the impact of denaturation and spray-drying on the exposition of these functional groups (Fig. 3). As expected, denaturation favored exposition of free thiol in both, solution and spray-dried powder forms. Unfolding of polymeric chains following denaturation was the main reason for the exponential increase in thiol groups [START_REF] Hsein | Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength[END_REF] whereas rearrangement of functional groups during particle formation probably decreased accessible thiol groups following spray-drying.

Rearrangement could be the reformation of aggregates by thiol-disulfide interchange [START_REF] Carrasquillo | Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein[END_REF] or the sharing of total free thiol groups between the surface and the core of particles.

Despite the mentioned decrease, the amount of accessible free thiol remained very high after denaturation compared to the amount of thiol sufficiently effective to exhibit functional properties such as mucoadhesion and bioavailability enhancement as described for other thiomers [START_REF] Ijaz | Bernkop-Schnürch, Preactivated thiomers: their role in drug delivery[END_REF][START_REF] Bonengel | Thiomers -From bench to market[END_REF].

Wettability

Contact angle between a liquid droplet and a flat surface is a direct indicator of the surface wettability. Value less than 90° characterize good wettability properties. The two explored polymers showed moderate to good wettability (<90°) with two different behaviors. For NWP-sp, contact angle was around 50 ± 2°, which is in agreement with published data [START_REF] Gaiani | How surface composition of high milk proteins powders is influenced by spray-drying temperature[END_REF].

Denaturation significantly increased contact angle to reach 82 ± 2°. Though, denaturation decreased WP wettability, which could be correlated to the exposure of hydrophobic groups following chain unfolding induced by denaturation.

Solubility

Whey protein structure are known to be thermosensitive and prone to solubility changes following denaturation [START_REF] Pelegrine | Whey proteins solubility as function of temperature and pH[END_REF] but no information is available about these properties following atomization. Unrefined WP were highly soluble with pH dependency, solubility increasing with pH (Table 2), in agreement with solubility values determined by Pelegrine and Gasparetto [START_REF] Pelegrine | Whey proteins solubility as function of temperature and pH[END_REF]. Spraydrying of NWP did not significantly affect solubility indicating the absence of protein degradation through denaturation during atomization in the selected operating conditions [START_REF] Anandharamakrishnan | Loss of solubility of α-lactalbumin and βlactoglobulin during the spray drying of whey proteins[END_REF].

On the contrary, denaturation induced a sharp decrease in DWP-sp solubility irrespective of the pH (Table 2). Insolubility of DWP-sp is due to conformational changes of proteins resulting from unfolding and exposure of hydrophobic and thiol groups leading to hydrophobic and disulfide intermolecular bonds [START_REF] Frank | Effect of pH and temperature on protein unfolding and thiol/disulfide interchange reactions during heat-induced gelation of whey proteins[END_REF][START_REF] Donovan | Thermal Denaturation and Aggregation of Whey Proteins[END_REF]. These interactions are responsible for intermolecular aggregation as shown in FTIR spectra resulting in a cross-linked insoluble network [START_REF] Mchugh | Plasticized Whey Protein Edible Films: Water Vapor Permeability Properties[END_REF]. Insolubility of DWP-sp was a required factor for further applications such as controlled release of encapsulated drugs and can represent a promising advantage as a carrier to avoid fast release.

Biopharmaceutical properties of spray-dried DWP powders

Strategies developed to improve drug bioavailability are mainly based on excipient biopharmaceutical properties. These strategies include: (i) prolonging gastro-intestinal residence time by choosing mucoadhesive excipient, (ii) protecting API from enzymatic degradation by using enzymatic inhibitory and (iii) promoting paracellular transport by using absorption enhancers [START_REF] Yun | Controlled Drug Delivery: Historical perspective for the next generation[END_REF]. In addition to these strategies, controlled release is of great importance since it allows improving drug efficacy, reducing toxicity, and improving patient compliance and convenience [START_REF] Smart | Oral peptide and protein delivery: intestinal obstacles and commercial prospects[END_REF].

Apparent dissolution

Apparent dissolution profiles of NWP-sp and DWP-sp powders were evaluated in a USP 4 closed system, at pH 1.2 and at pH 6.8, by monitoring the release of WP (Fig. 4). NWP-sp exhibited very fast dissolution with 70% released in 5 min whatever the pH. At pH 6.8, dissolution was complete but at pH 1.2, dissolution didn't exceed 75%. Incomplete dissolution despite working in sink conditions was probably due to the moderate wettability properties [START_REF] Gaiani | Dairy powder rehydration: influence of protein state, incorporation mode, and agglomeration[END_REF][START_REF] Kim | Surface characterization of four industrial spray-dried dairy powders in relation to chemical composition, structure and wetting property[END_REF] of NWP-sp and to the hydrodynamic conditions. In flow-through cell dissolution, flow is laminar and shear is provided by the pulsatile flow over the particles [START_REF] Gao | In Vitro Dissolution Testing with Flow-Through Method: A Technical Note[END_REF].

Gentle laminar flow may cause the deposit of a thin layer of particles on the glass beads, so that, in the case of NWP-sp, aggregation may occur and thus reduce the extent of dissolution. Denaturation modified completely the WP dissolution profile: DWP-sp dissolution was zero at pH 1.2. We have to note that it was impossible to work in sink conditions because its solubility was far too low (approx. 2 µg.mL -1 ) regarding experimental conditions. At pH 6.8, DWP-sp exhibited a zero-like order dissolution rate.

This low release rate correlates with the solubility study and is promising for drug controlled delivery.

Mucoadhesive properties

Mucoadhesive behaviors of spray-dried powders were assessed by measuring adhesion of particles to isolated rat intestinal fragments after 1 h: mucoadhesivity of NWP-sp was low (19.3 ± 2.9%) whereas DWP-sp exhibited high mucoadhesion to the intestine (48.7 ± 4.5 and 40.9 ± 3.4%, respectively). Though, denaturation favored mucoadhesive properties of WP-sp powders. This was due to unfolding of polymeric chains following denaturation, which expose functional groups such as free thiols and hydrophobic groups. Thiol functional groups are the main key for mucoadhesion because they allow establishment of covalent disulfide bonds between polymer and cysteines of the mucus [START_REF] Hsein | Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength[END_REF]. By comparison with mucoadhesivity of hydrogel or microparticles made of DWP [START_REF] Hsein | Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength[END_REF][START_REF] Withers | Investigation of milk proteins binding to the oral mucosa[END_REF], these results effectively proved that the denaturation clearly exhibited these properties and the spray-drying technique turned to maintain them.

Mucoadhesivity of DWP-sp, especially compared to NWP-sp, render them to be good carriers for drug administration because mucoadhesion prolong residence time and facilitate intimate contact with the intestinal mucosa playing though an important role in enhancing bioavailability [START_REF] Andrews | Mucoadhesive polymeric platforms for controlled drug delivery[END_REF].

Enzymatic inhibitory effect

The protective property of a polymeric delivery system towards enzymatic degradation is primordial in case of oral administration of enzyme-sensitive drug like therapeutic proteins. Amongst luminally secreted proteases and membrane bound proteases, trypsin and chymotrypsin are the most abundant secreted proteases with the highest enzymatic activity and they were though chosen as model proteases to study the inhibition effect of WP [START_REF] Bernkop-Schnürch | The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins[END_REF].

Both NWP-sp and DWP-sp powders inhibited enzymatic activity (Fig. 5). Nevertheless, the efficacy of DWP-sp to inhibit the enzymatic action was significantly superior to NWP-sp (Fig. 5). Being a protein moiety, WP can be considered as a substrate for proteases and thus can play the role of inhibitor as observed with the commonly used Bowman-Birk inhibitor extracted from soybean protein [START_REF] Birk | The Bowman-Birk inhibitor. Trypsin-and chymotrypsin-inhibitor from soybeans[END_REF].

Cytotoxicity studies and paracellular transport enhancement

Cytotoxicity study was carried on by the blue trypan exclusion test which indicates membrane integrity [START_REF] Strober | Trypan Blue Exclusion Test of Cell Viability[END_REF]. None of the tested polymers induced cell cytotoxicity: more than 90% viable cells were counted after 2 h incubation (96.54 ± 2.33%, 93.07 ± 3.65% and 91.52 ± 4.18% for NWP-sp and DWP-sp, respectively). So, DWP-sp, by comparison to other calcium chelating agent used as enzymatic inhibitor (EDTA, EGTA [START_REF] Bernkop-Schnürch | The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins[END_REF][START_REF] Touitou | Enhancement in Drug Delivery[END_REF]) , doesn't present any toxic effect and though can be considered more advantageous for drug delivery.

The effect of spray-dried powders on the paracellular transport of PEG 4000 was assessed.

The transport of this model compound is known to be through tight junction opening due to its hydrophilic and high molecular weight characters [START_REF] Shah | Role of 99mTc-mannitol and 99mTc-PEG in the assessment of paracellular integrity of cell monolayers[END_REF].

At the beginning of experiments, all Caco-2 monolayer cells presented TEER values greater than 600 Ω.cm 2 indicating the integrity of the tight junctional complex. In comparison to untreated cells and to cells treated with NWP-sp, addition of DWP-sp powders to the apical side induced a significantly decrease in TEER values and an increase in the Papp of PEG 4000. TEER decreased to reach 60% of its initial value after 2 h incubation (Fig. 6). At the tight junctional complex, TEER values are usually reduced due to water and ion passage through the paracellular route [START_REF] Sambuy | The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics[END_REF]. Thus, DWP-sp powders seemed to exert an effect on paracellular permeability by opening tight junctions. This hypothesis was confirmed by the two time increased P app of PEG 4000 (Table 3). Similar results were obtained by Déat-Lainé et al. [START_REF] Déat-Lainé | Whey protein and alginate hydrogel microparticles for insulin intestinal absorption: evaluation of permeability enhancement properties on Caco-2 cells[END_REF], evaluating the effect of DWP polymeric solutions and microparticles on paracellular passage of insulin on Caco-2 cells. Thus, spray-drying preserved well DWP properties to enhance paracellular transport and could be thus, an interesting alternative to produce dried DWP particles in a single-step.

The ability of Caco-2 monolayers to recover to their original impermeable state after sample removal was evaluated by monitoring TEER values for up to 24 h. For all samples, TEER values returned to their baseline levels in 24 h. Consequently, as previously demonstrated for DWP microparticles [START_REF] Déat-Lainé | Whey protein and alginate hydrogel microparticles for insulin intestinal absorption: evaluation of permeability enhancement properties on Caco-2 cells[END_REF], DWP-sp powder can be considered as a good excipient to temporary modulate paracellular permeability and to promote drug absorption without toxic effect.

Conclusion

High yield and low residual humidity unloaded DWP-sp particles, with almost perfect spherical forms, were obtained in our study using spray-drying method. DWP-sp powder exhibited the characteristics required to increase bioavailability of a drug after oral administration.

Compared with native one, DWP-sp showed an unfolding of polypeptide segments with intermolecular β-sheets resulting from aggregation. The free thiol exposure favored by denaturation was preserved after drying, and thus the DWP-sp powder was mucoadhesive at intestinal pH due to possible establishment of covalent disulfide bridges between mucin and polymer.

The obtained powder of DWP-sp, known to be calcium chelating agent, exhibited a protective inhibitory activity effect against proteases; which is a requirement for therapeutic peptide/protein oral administration. Moreover, this powder had no cytotoxic effect on Caco-2 cells and promoted reversible opening of tight junction, which allow increasing drug absorption by paracellular route.

In addition, dissolution of DWP-sp showed interesting slow release properties to push forward investigations concerning impact on drug controlled release for oral delivery.

In view of the observed physico-chemical and biopharmaceutical properties, manifested in our study, spray drying is a single step technique for preparing DWP based particles to operate as excipients for modifying the dissolution rate of drugs and enhancing the bioavailability of molecules, such as therapeutic proteins.

Fig. 1 .Fig. 2 .

 12 Fig. 1. SEM images (x3500) for spray-dried WP particle samples: (a) NWP, (b) DWP (10% WP content, inlet temperature 160°C, pump rate 5.7 mg.min -1 and Q-flow 50 mmHg).

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. Comparison of free thiol concentrations in liquid and in spray-dried samples: influence of denaturation and glycerol addition (n=3, mean ± SD, * and † p <0.05 significantly different from solution of NWP and DWP

Fig. 6 .

 6 Fig. 6. TEER (%) values obtained on Caco-2 cells after 2 h incubation of NWP, DWP and DWP-Gly powders on Caco-2 cells (n = 3, mean ± SD).

Table 1 .

 1 Spray-drying of WP solutions (10%, w/w): Evaluation of response variables (yield, residual humidity, outlet temperature and particle size) as a function of applied process parameters (inlet temperature 160°C, pump rate 5.7 mg.min -1 and Q-flow 50 mmHg) (n=3 ± SD).

	WP form	NWP	DWP
	Yield (%)	87.3 ± 0.1	80.8 ± 0.4
	Residual humidity (%)	4.5 ± 0.2	3.9 ± 1.2
	Outlet temperature (°C)	88.6 ± 1.2	72 ± 1.0
	D [3,2] µm	4.2 ± 2.6	4.2 ± 2.7

Table 2 .

 2 Solubility (mg.mL -1 ) of WP powders (unrefined WP, NWP-sp or DWP-sp) in pH 1.2, 4.5, 6.8 or 7.5 buffers (n = 3, mean ± SD).

	Buffer	pH 1.2	pH 4.5	pH 6.8	pH 7.5
	Unrefined WP	502.6 ± 30.8	772.5 ± 51.2	835.2 ± 47.2	872.5 ± 39.7
	NWP	469.8 ± 26.9	723.4 ± 38.5	784.4 ± 33.8	867.6 ± 33.3
	DWP	2.2 (± 0.3).10 -3	8.8 ± 0.3	14.4 ± 2.1	16.9 ± 1.4

Table 3 .

 3 Mean apparent permeability (P app ) of PEG 4000 and absorption enhancement ratio (R) across Caco-2 cell monolayers, alone (control, free PEG) or with NWP-sp or DWP-sp powder after 2h incubation (n = 3, mean ± SD, * p <0.05 significantly different from control).

	Sample	Absorption		P app	R
		µg	%	x (10 -6 .cms -1 )
	Control	52.96 ± 2.55	11.21 ± 0.54 6.95 ± 0.03	1.00
	NWP	47.72 ± 2.40	10.36 ± 0.52 6.42 ± 0.03	0.92
	DWP	100.44 ± 5.02	21.80 ± 1.09 13.20 ± 0.06* 1.91*


Stefano Manfredi

The construction of linguistic borders and the rise of national identity in South Sudan: some insights into Juba Arabic (árabi júba)

This paper explores the nexus between the social construction of linguistic borders and the rise of a national identity in post-independence South Sudan. It analyses the indexical functions of Juba Arabic (i.e. árabi juba), an Arabic-based pidgincreole extensively exposed to contact with Sudanese Arabic, its lexifier and former dominant language. By combining insights from descriptive linguistics and discourse analysis, I will argue that, although the metalinguistic representations of Juba Arabic are increasingly driven by the ideological hegemony enacted by the State through its discourse on "indigenous languages", the construction of linguistic borders between the South Sudanese pidgincreole and its lexifier can also be constrained by cognitively prominent grammatical features.

1.

Introduction Until nowadays, two theoretical paradigms have dominated to describe creolelexifier language contact [START_REF] Goury | Contacts de créoles, créoles en contact. Présentation[END_REF]. The first one is the Fergussonian paradigm of diglossia, which describes creole-lexifier contacts as stable sociolinguistic situations involving two linguistic varieties with specific functional assignments [START_REF] Ferguson | Diglossia[END_REF][START_REF] Carayol | Diglossie et continuum linguistique à la Réunion[END_REF]. The second one is decreolization, which postulates an asymmetric contact producing the gradual replacement of creole features by those of its lexifier along the lines of a unidirectional continuum running from two poles, the basilect and the acrolect, through a series of mesolectal varieties (De Camp 1971;[START_REF] Bickerton | On the nature of a creole continuum[END_REF]. Different from diglossia that focuses on functional variation and societal norms, the notion of decreolization refers back to the structural effects of a process of attrition leading to language shift. Despite its relative success, the decreolization paradigm has been repeatedly criticized during the last decades. Above all, it has been argued that decreolization does not tell anything about variation induced by internal factors [START_REF] Michael | Looking beyond decreolization as an explanatory model of language change in creole-speaking communities[END_REF][START_REF] Mufwene | Language and the Social Construction of Identity in Creole Situations[END_REF]). Furthermore, being essentially descriptive, decreolization is unable to reflect language attitudes and ideologies lying behind creole-lexifier language contacts [START_REF] Migge | On the emergence of new language varieties: the case of the Eastern Maroon Creole in French Guiana[END_REF]. Lastly, the fact that decreolization is often analyzed in the light of a (post-)creole continuum represents a vexing problem for linguists since it is extremely difficult to draw implicational scales reflecting the gradual shift towards the lexifier language. This is because each speaker tends to have its own way of mixing creole and lexifier features, some converging more on phonological features, others on morphology and/or lexicon [START_REF] Miller | Do they speak the same language? Language use in Juba local courts[END_REF]. Irrespective of this epistemological problem, (post-)creole continua are generally assumed to be different from dialect continua in that the basilectal and acrolectal poles are typologically dissimilar to such an extent that they are considered to be distinct languages [START_REF] Siegel | Decreolization: a critical review[END_REF].

In this last regard, a common assumption of post-modern sociolinguistics is that linguistic differentiation always involves ideologically embedded and socially constructed processes [START_REF] Irvine | Language ideology and linguistic differentiation[END_REF]. This is the core point of view of globalization studies which postulate that named languages are ideological constructions tied to the emergence of Nation-States and that there are not truly distinct linguistic systems bounded by grammars, as habitually argued by descriptive linguists [START_REF] Blommaert | Language and superdiversity[END_REF][START_REF] Jørgensen | Polylanguaging in superdiversity[END_REF]. In this framework, language borders in contact situations are conceived as means of social categorization, which are only relevant when the speakers deliberately constructs them [START_REF] García | Translanguaging: Language, Bilingualism and Education[END_REF]. It is not surprising, then, that most studies relativize the outcomes of the semiotic process of language differentiation by putting emphasis on the lack of coincidence between the linguistic borders constructed by speakers and those drawn by linguists [START_REF] Bert | What counts as a linguistic border, for whom and with what implications? Exploring Occitan and Francoprovençal in Rhône-Alpes, France[END_REF][START_REF] Cornips | Language contact, linguistic variability and the construction of local identities[END_REF].

It is against this background that the present study addresses the issue of the social construction of linguistic borders in a creole-lexifier contact situation. It focuses on Juba Arabic, an Arabic-based pidgincreole spoken in South Sudan [START_REF] Manfredi | Arabi Juba: un pidgin-créole du Soudan du Sud[END_REF]. Before the South Sudan independence in 2011, Juba Arabic has been extensively exposed to contact with Sudanese Arabic, the dominant language of the former unified Sudan. Thereafter, new contact situations between the pidgincreole and its lexifier emerged due to returnees entering South Sudan from Arabic dominant regions. This prolonged contact eventually led to different degrees of linguistic interference. The first to analyze Juba Arabic in terms of (post-)creole continuum was [START_REF] Mahmoud | Variation in the Aspectual System of Juba Arabic[END_REF][START_REF] Miller | Kelem kalam bitak: langues et tribunaux urbains en Equatoria[END_REF]2007), on her part, insists on the fact that, although affected by its lexifier, the evolution of the South Sudanese pidgincreole is far from being a linear continuum towards Sudanese Arabic. In the light of the above, the present study faces the question of whether Juba Arabic speakers argue for a differentiation between their language and Sudanese Arabic and, if it is the case, to what extent the construction of this linguistic border depends on local ideological factors or on the identification of relevant linguistic features.

As a matter of fact, due to their atypical conditions of emergence, pidgins and creoles are particularly relevant for the discussion of the ideological link between language and identity [START_REF] Edwards | Language and Identity[END_REF]. Being spoken as vehicular languages, pidgins do not function as identity markers. In contrast, nativized creoles may develop identity functions in relation to ethnic groups (e.g. the Arabic-based Ki-Nubi creole in Kenya and Uganda) or Nation-States (e.g. the French-based Haitian Creole). As far as Juba Arabic is concerned, it is a pidgincreole [START_REF] Tosco | Pidgins and Creoles[END_REF], that is an intermediary category defined by the fact that an earlier pidgin has become the first language only for a part of its speakers [START_REF] Bakker | Pidgins versus creoles and pidgincreoles[END_REF]. This means that, although Juba Arabic is largely used as vehicular language, it shares some sociolinguistic properties with creoles. Miller has already stressed that Juba Arabic functions as marker of a super-ethnic South Sudanese identity since the early 1970s [START_REF] Miller | Le changement linguistique à Juba et à Khartoum[END_REF][START_REF] Miller | Juba Arabic as a way for expressing a Southern Sudanese identity in Khartoum[END_REF]. More recently, the South Sudanese independence further straightened the identity functions of Juba Arabic. Accordingly, this study is also concerned with the question of which effects the rise of a South Sudanese national identity have on language categorization and on the construction of language borders.

The paper is organized as follows. Section 2 draws a background to language policies and language categorization in post-independence South Sudan. Section 3 deals with the definition of Juba Arabic from a descriptive perspective and it presents the main features used by linguists for distinguishing between Juba Arabic and its lexifier. Section 4 then analyses the linguistic borders constructed by speakers in relation to both prominent grammatical features of Juba Arabic and local ideological factors. Section 5, finally, investigates the nexus between speakers' attitudes towards Juba Arabic and the rise of a South Sudanese national identity.

2.

Background : language policies and language categorization South Sudan presents a high degree of linguistic diversity, with about seventy languages belonging to three out of the four language families attested in Africa (i.e. Nilo-Saharan, Niger-Congo and Afro-Asiatic). In this complex linguistic landscape, the Arabic-based pidgincreole called árabi juba (i.e. Juba Arabic) represents the first means of interethnic communication of the country, while being nativized by the largest part of the urban population of Juba, the capital city of South Sudan (Manfredi and Tosco forth.).

Following the Sudan's comprehensive peace agreement in 2005, South Sudan moved from a strict Arabic monolingualism to a policy of multilingualism. In 2011, English has been recognized as official language, whereas all the "indigenous" languages of the country were accorded the status of national languages. What really counts as "indigenous language" is obviously difficult to say, since this category can only be defined in opposition to an "alien language", in most cases represented by a colonial language. The fact remains that Juba Arabic is officially considered to be a variety of Arabic, and therefore it is not recognized as an "indigenous language" of South Sudan (Manfredi and Tosco forth.). Most importantly, being a vehicular language, Juba Arabic does not fit within the ethnic understanding of "indigenous languages" inherited from the colonial language policy and renewed by the South Sudan independence (Abdelhay 2007;[START_REF] Abdelhay | The Naivasha language policy: The language of politics and the politics of language in the Sudan[END_REF]. Despite this, I will show that the institutionalization of "indigenous language" as a sociolinguistic category has a significant impact on the social categorization of Juba Arabic.

As a further matter, folk Arabic-centric linguistic ideologies are still at work in post-independence South Sudan and these also affect language categorization. The enduring semiotic opposition between the labels luġa and ruṭāna is a case in point. The Arabic term luġa (lúga in Juba Arabic) means "language" and has a normative value in Sudan. It refers back to a high prestige linguistic variety, with an established written tradition and wide community of speakers. luġa/lúga is primarily used in relation with Arabic, but it can also label European languages and other languages of wider communication. In contrast, the Sudanese Arabic term ruṭāna (rután in Juba Arabic) can be broadly translated as "incomprehensible (ethnic) language; patois" and it conveys a marked (depreciative) value. It refers to low-prestige languages, with no written tradition and spoken by small ethnic groups. Sudanese Arabs usually adopt the term ruṭāna for labelling all the non-Arabic linguistic entities present in the region. 1The analysis of the interviews in sections 4 and 5 shows that Juba Arabic occupies an ambiguous position within the traditional luġa / ruṭāna semiotic dichotomy. However, since discursive phenomena are always hierarchically ranked [START_REF] Blommaert | Sociolinguistic scales[END_REF]), I will illustrate the different intertextual meanings of the terms luġa / lúga, ruṭāna / rután, and indigenous language when used for categorizing Juba Arabic and/or Sudanese Arabic. These considerations will turn out to be useful when analyzing the construction of a linguistic border between the South Sudanese pidgincreole and its lexifier.

3.

Is Juba Arabic different from Arabic? The linguists' perspective This section aims at illustrating the main features used by linguists for establishing a border between Juba Arabic and its lexifier. These will furnish a comparative basis for the analysis of the metalinguistic representations expressed by ordinary speakers of Juba Arabic in section 4.

Creole languages are considered to be different from other spoken languages in that they came into existence as a consequence of the disruption of the intergenerational transmission of the lexifier language from parent to child [START_REF] Comrie | Creoles and language typology[END_REF]. These uncommon conditions of language emergence entail different processes of language change linked to second language acquisition, substratum interference as well as to internal developments. Having emerged in a situation of second language acquisition with limited access to the target language, Juba Arabic presents typical features of creole languages [START_REF] Tosco | Pidgins and Creoles[END_REF].

First of all, Juba Arabic draws about 80% of its lexicon from its Arabic lexifier [START_REF] Manfredi | Arabi Juba: un pidgin-créole du Soudan du Sud[END_REF]. The remaining 20% is composed by an increasing number of loanwords from Nilotic languages whose integration mostly affects the non-basic (i.e. culturally marked) lexicon of the pidgincreole. Most loanwords are from Bari, the main substrate/adstrate language of Juba Arabic; these are either nouns (e.g. lógoro "heron") or verbs (e.g. kúruju "cultivate", Nakao 2012). We also find a number of borrowed nouns from European languages (mainly from English, e.g. síster "nun"; nígas "street boy") as well as from other languages of wider communication like Swahili (e.g. wéwe "East African migrant"). All things considered, lexicon is certainly not the most relevant domain for establishing a linguistic border between Juba Arabic and Sudanese Arabic.

The phonology of Juba Arabic, on its part, has been forged by a process of second language acquisition in combination with an important substrate interference. On the one side, Juba Arabic is characterized by the loss of Arabic complex realizations which are absent in the Nilotic substrate (i.e. velar fricatives x and ġ; pharyngeal fricatives ḥ and '; all the pharyngealized consonants ṭ, ḍ; ṣ etc.). On the other side, it presents non-Arabic complex realizations entered from substrate/adstrate languages via lexical borrowing (e.g. the voiced bilabial implosive in b'ondó "village" and the voiced alveolar implosive in d'éŋele "bile"). Furthermore, Arabic stress has been reinterpreted as a pitch accent in Juba Arabic. Phonology thus provides a good linguistic evidence for distinguishing between Juba Arabic and Sudanese Arabic. Nevertheless, other vehicular varieties of Arabic spoken by minority groups in the Arabic-dominant regions of Sudan and South Sudan display similar phonological features [START_REF] Manfredi | Native and non-native varieties of Arabic in an emerging urban centre of Western Sudan. Evidence from Kadugli[END_REF], ant these are not the product of a process of pidginization/creolization as in the case of Juba Arabic.

Turning to morphosyntax, Juba Arabic is characterized by an important reduction of the inflectional and derivational categories of Sudanese Arabic. More generally, an important typological difference between Juba Arabic and Sudanese Arabic resides in the isolating morphology of the pidgincreole as compared to the fusional morphology of its Arabic lexifier. In this regard, Versteegh (1993: 73) affirmed that "agreement markers in the verbal system of Juba Arabic is of the utmost importance for the comparison between creole and "normal" (Arabic) dialects". The following table corroborates this view.

Table 1. Paradigm types

Baggara Arabic (Kadugli, Sudan) (Manfredi 2010: 129) bi=na-mš-u IND=1-go-PL "We (will) go" Vehicular Arabic (Kadugli, Sudan) (Manfredi 2014: 30) na-mši 1-go "I (will) go, we (will) go" Juba Arabic (Juba, South Sudan) (Manfredi 2017: 83) ánna bi=rówa 1PL IRR=go "We (will) go"

As we can see, the Sudanese lexifier, here exemplified by the Baggara dialect of Kordofan, presents a typical Arabic imperfective paradigm with personal affixes indexing aspect, number and gender, and a proclitic marking the indicative mood on the verb. In contrast, in Juba Arabic, the subject is indexed by an independent pronoun preceding an invariable verbal form marked for irrealis. In this respect, Juba Arabic is also different from other vehicular varieties of Arabic that, though affected by some degree of morphological simplification (as showed by the conflation of 1 st singular and plural persons), do not display the same degree of restructuring of the South Sudanese pidgincreole.

It is important to remind that Versteegh, basing his argument on the variationist study of [START_REF] Mahmoud | Variation in the Aspectual System of Juba Arabic[END_REF], has argued that Juba Arabic is increasingly integrating personal affixes from Sudanese Arabic and, consequently, "we can envisage a future in which the linguistic varieties spoken in Juba become more and more similar to Khartoum Arabic. […] In the end, this scenario might lead to a situation where Juba Arabic would be nothing more than a regional variety of Sudanese Arabic." (Versteegh 1993: 75). Almost twenty years after this prediction of direction-of-change, we can affirm that it did not come true, since the bulk of Juba Arabic speakers still present typical creolized verbal paradigms [START_REF] Manfredi | Arabi Juba: un pidgin-créole du Soudan du Sud[END_REF]. This fact may find a reason in the ideological resilience to integration of highly identifiable linguistic items from Sudanese Arabic. The analysis of interviews in section 4 indeed shows that verbal morphosyntax is a cognitively relevant linguistic feature for the construction of a border between Juba Arabic and Sudanese Arabic.

Is Juba Arabic different from Arabic? The speakers' perspective

In this section, I shall investigate the metalinguistic representations that Juba Arabic speakers express about their language based on the analysis of qualitative interviews recorded in Juba in July -August 2013 2 . The aim of the analysis is to individuate the linguistic features used by speakers for indexing Juba Arabic vis-à-vis its lexifier and to compare them with those features that are meaningful to linguists (cf. 3).

When asked whether there is any linguistic differences between the South Sudanese pidgincreole and its lexifier, Juba Arabic speakers who do not speak Sudanese Arabic broadly recognize the existence of a linguistic border, without referring to any particular features as we can see in the following excerpt of interview 3 . Excerpt 1. Speaker 1 4 fi férig kebír // mar-át ána ma gi=fáhim // kan bíga / úmon bi=dákal fi árabi ta kartúm de / ána ma bi=fáhim kúlu kúlu // úmon kamán ma gi=fáhim árabi tái // úmon bi=kélim gal / hája de rután // "There is a huge difference. Sometimes I do not understand. If they start speaking Khartoum Arabic, I do not understand at 2 Fieldwork in Juba (South Sudan) was carried out by Stefano Manfredi and Mauro Tosco (University of Turin) and it was made possible by a grant from the Italian Ministry of Education, University and Research (MIUR) within the project "Areas of Linguistic and Cultural Transition in Africa" (ATrA). 3 The interviews used for this study mainly focused on the metalinguistic representations and language attitudes towards Juba Arabic (see also Manfredi and Tosco forth.). The interviews were conducted in Juba Arabic, English and Sudanese Arabic. The following excerpts of interviews are coupled with metadata relating to each speaker. These include sociolinguistic information such as gender, age, place of birth, degree of formal education, patterns of bi/multilingualism and, when relevant, migration trajectories. Speech chunks in Juba Arabic, Sudanese Arabic and Modern Standard Arabic are phonologically transcribed, whereas English is orthographically transcribed. Juba Arabic is reproduced in italic; Sudanese Arabic and Modern Standard Arabic are reproduced in bold, and English is underlined. All the excerpts display both morphological and prosodic segmentation (= and -respectively mark clitic and affix boundaries, // and / mark major and minor prosodic boundaries, ↑ indicates a rising intonation). 4 Male, 25 years old, born in Yei, primary education, L1 Juba Arabic, L2 Pojulu, moved to Juba in 1995.

all. They also do not understand my Arabic. They would say: this is a rután."

The view articulated by this young speaker is that Juba Arabic and Sudanese Arabic are distinct languages. His empirical assumption is based exclusively on the low degree of mutual intelligibility experienced by the speakers of the two languages. This factual reality is also acknowledged by linguists [START_REF] Manfredi | Arabi Juba: un pidgin-créole du Soudan du Sud[END_REF]. More interestingly, in order to stress the perceived linguistic distance, the interviewee evokes the depreciative term rután as used by Sudanese Arabic speakers when referring to Juba Arabic. Thus, in this case, rután does not give evidence of the speaker's semiotic processing of sociolinguistic categories. It rather reproduces the downscaling of Juba Arabic operated in conformity to the Sudanese Arabic-centric layered system, opposing a high prestige variety (i.e. Sudanese Arabic) labelled as luġa "language" to a number of low prestige "incomprehensible (ethnic) languages" (cf. 2).

In contrast, Juba Arabic speakers who also speak Sudanese Arabic tend to construct linguistic borders constrained by specific linguistic features as we can see in the following excerpt of interview.

Excerpt 2. Speaker 25 (Manfredi and Bizri, forth.) úo ma árabi // [...] zey hása fi šamál bi=nadí gal / luġa 'arabiyya // árabi júba kamán / gi=nadí gal lúga // zey ána bi=kélim gal / ána kélim le íta // lakín úmon henák / úmon bi=kélim gal / b=a-gūl lē=k // "(Juba Arabic) is not (Sudanese) Arabic. Like, for example, in the north it is called luġa 'arabiyya ("Arabic language"). Juba Arabic too is called lúga. Like, I say: ána bi=kélim le íta ("I say to you"). But, over there, they say: b=a-gūl lē=k ("I say to you")."

This trilingual speaker uses the normative term luġa / lúga for referring to both Sudanese Arabic and Juba Arabic. 6 He thus operates an upscaling in the local system of sociolinguistic categories. This means that he accords Juba Arabic the same linguistic prestige as its lexifier language and considers it as a fullfledged "language". Besides, the speaker constructs a linguistic border between Juba Arabic and Sudanese Arabic by opposing two verbal clauses; these are ána bi=kélim le íta, 1SG IRR=say to 2SG, "I say to you" in Juba Arabic and b=a-gūl lē=k, IND=1SG-say to=2SG.M, "I say to you" in Sudanese Arabic. Regardless the different lexical verbs conveying the meaning of "(to) say", we can note that, in the Juba Arabic example, the verb lacks personal affixes and both the subject and the indirect object are encoded by independent pronouns. Conversely, in the Sudanese Arabic example, the subject and the indirect object of the verbal clause are respectively encoded by a person affix on the verb and a clitic pronoun attached to the preposition lē "to". What makes this excerpt particularly interesting is that the factual-descriptive border constructed by the speaker clearly corresponds to the linguists' representation of Juba Arabic as an isolating language (cf. 3).

The third excerpt of interview also confirms that verbal morphosyntax is a cognitively prominent feature for the identification of Juba Arabic. Excerpt 3. Speaker 3 7 másalan / for example // ána kan gi=wónus kílma // másalan / ána gi=rówa // aw ánna gi=fútu // de árabi juba // for example / kan zol min kartúm gal // mašē-na // aw / mašē-t // ána tawáli b=ágder árif kalám de took rise min zol al ja min kartúm // aw kan gi=géni fi kartúm ketír // so on and so forth // 6 In the first occurrence of the term, we can observe a code-mixed sentence corresponding to a prosodically isolated instance of direct speech including the two adjacent elements luġa 'arabiyya "Arabic language" which are both drawn from Arabic, as testified by the presence of the voiced velar fricative /ġ/ and the voiced pharyngeal fricative /'/. In the second occurrence, the realization of the term lúga conforms to the phonological rules of Juba Arabic (i.e. absence of the voiced velar fricative /ġ/) and it is not prosodically marked. 7 Male, 31 years old, born in Juba, secondary education, L1 Juba Arabic, L2 English, L3 Sudanese Arabic.

"For example, if I say, for example: ána gi-rówa ("I am going"), or ánna gi=fútu ("we are passing"). This is Juba Arabic. For example, if someone from Khartoum says: mašē-na ("we went"), or mašē-t ("I went"), I can immediately know that this discourse took rise from someone who came from Khartoum, or who spent a long time in Khartoum, so on and so forth."

This interviewee too gives a factual representation of Juba Arabic based on the opposition between different verbal paradigms. These are ána gi=rówa ("I am going") and ánna gi=fútu ("we are passing"), corresponding to the perceived morphological norm of Juba Arabic, and mašē-na ("we went") and mašē-t ("I went"), representing the deviated Arabic-like verbal paradigms. In this particular case, the speaker operates a process of iconization [START_REF] Irvine | Language ideology and linguistic differentiation[END_REF] when considering the occurrence of person affixes on the verb as formally congruent with returnees "who spent a long time in Khartoum". In so doing, the interviewee construct a community-internal linguistic border between South Sudanese returnees, who have evidently been much exposed to Sudanese Arabic, and South Sudanese long-term residents, who are perceived to be the norm holders.

Juba Arabic speakers may also focus on lexical features for distinguishing between the pidgincreole and Sudanese Arabic, as exemplified by the following excerpt of interview.

Excerpt 4. Speaker 4 8 úo ma árabi // [...] de rután adíl // de rután baráu // úo / of course / ána árif úo ja min árabi // wo borrowed kalim-át min rutan-át / local // rutan-át / bári / rután šunú šunú / wo biga mulágbat // le dérija / árabi / árabi ma gi=fáhim // you know // árabi ma gi=fáhim // "(Juba Arabic) is not (Sudanese) Arabic. This is a rután, a rután on its own. It, of course, I know that it derived from Arabic and that it borrowed words from rutan-át, local, rutan-át such as Bari, whatever rután, and it became mixed to such extent that Arabs do not understand. You know, they do not understand."

The view articulated by this speaker is that, even if Juba Arabic clearly derives from (Sudanese) Arabic, it is a distinct language to such an extent that "Arabs do not understand it". He then refers to the South Sudanese creole with the term rután. Different from excerpt 1, here rután does not convey a depreciative value, but it rather points out the centrality of the category "local" in the discourse regarding Juba Arabic. Not surprisingly, the term rután is coupled with a metalinguistic representation concentrating on the lexical contribution of "local" languages to Juba Arabic. This view is further strengthened by the following excerpt of interview.

Excerpt 5. Speaker 5 9

Juba Arabic is different // it has been indigenized in some way // some tribal words of the indigenous languages / in the south / have gone inside // [...] such as Bari / b'ángiri / kúrju // So you can see / be taríga de / árabi júba šílu / yáni / šikil /šikil / afrígi // [...] laánu kalim-át al=maḥalliyya / dákalu fógo ketír // "Juba Arabic is different. It has been indigenized somehow. Some tribal words from South Sudanese indigenous languages have been integrated. Such as Bari, b'ángiri ("cheek"), kúruju ("cultivate"). So you can see, in this way, Arabic took an African shape. Because many maḥalliyya ("indigenous") words have gone inside."

This highly-educated interviewee overtly argues that the linguistic difference between Juba Arabic and Sudanese Arabic resides in the "indigenized" nature of pidgincreole. In his perspective, Juba Arabic is a language with an "African" shape presenting "tribal" words borrowed from South Sudanese "indigenous languages". This view confirms that, though rare (cf. 3), substrate/adstrate loanwords in Juba Arabic have a strong iconic function in relation to the official notion of "indigenousness". When the speaker portrays Juba Arabic as an "indigenous language" (using also the Arabic term maḥalliyya "indigenous"), he operates an upscaling in relation to State's prescriptions of the sociolinguistic regime. In other words, he attempts to legitimize Juba Arabic within the sociolinguistic space of post-independence South Sudan while accepting without any criticism the notion of "indigenous language".

Language attitudes and the rise of a South Sudanese national identity

While the previous section dealt with metalinguistic representations and the construction of linguistic borders, the last part of the study analyses language attitudes, here defined as beliefs and values that people have about their language [START_REF] Garret | Attitudes to Language[END_REF], in relation with the rise of a South Sudanese national identity.

Most Juba Arabic speakers have a positive attitude towards the South Sudanese pidgincreole. This general opinion goes hand in hand with the appreciation of the vehicular function of Juba Arabic in the multilingual landscape of South Sudan. Excerpt 6. Speaker 6 10 árabi júba lúga / ašán bi=límu jenúb kúlu // for example / kan íta rówa fi kénya / lúga tómon wáhid / yawú swahíli // swahíli bi=límu kúlu // nína fi jenúb sudán / hája al bi=límu nína kúlu / árabi júba // yála rután de bíga ta gebíla táki // kan íta abukáya / íta bi=wónusu abukáya // lakín árabi júba de ma gi=derisú // bes fi bét / árabi júba gi=wonusú / zey rután // lakín úo hása bíga lúga // "Juba Arabic is a lúga, because it bonds the whole South (Sudan). For example, if you go to Kenya, they have one language, that is Swahili. Swahili bonds everyone. As far as we are concerned in South Sudan, the thing that bonds us is Juba Arabic. The rután is something that belongs to your tribe. If you are Abukaya, you speak Abukaya. But Juba Arabic is not taught. It is spoken only at home, like a rután. However, it became a lúga."

In the previous excerpt, a young trilingual girl labels Juba Arabic as lúga. This upscaling in the local categorization of languages finds a reason in the emphasis putted on the unifying role of Juba Arabic in South Sudan. This view is reinforced by comparing the vehicular function of Juba Arabic with that of Swahili, and by contrasting it to the category rután, which is perceived as inevitably linked to single tribal entities. Furthermore, when the speaker states that, even if Juba Arabic became a lúga, it is acquired at home like a rután, she gives evidence of the perception of the ongoing nativization process of Juba Arabic.

The following excerpt of interview also confirms that the vehicular function of Juba Arabic is perceived as an important element for the achievement of South Sudanese national unit.

Excerpt 7. Speaker 5 Juba Arabic as a lingua franca / is the language that has a future in this country // it is a uniting bond that can bring South Sudanese together // because no one can claim dominating the other through this language // no Dinka would say / it is ours // [...] so it encompasses the whole South (Sudan) / as an important language that can equally go to primary schools / to / to basic level schools // Here, the speaker who previously emphasized the "indigenous" nature of the South Sudanese pidgincreole in excerpt 5, rather stresses that Juba Arabic is the only unifying Lingua Franca of South Sudan and, because of this, it cannot be instrumentalized in the light of the official ethnic-oriented understanding of language diversity. In the speaker's view, a non-ethnic language like Juba Arabic can also be "indigenous" and, as such, it should be recognized as national language and taught in primary schools.

In this context, it should be remarked that the glottonym árabi júba still hinders the adoption of Juba Arabic as a marker of the new South Sudanese national identity. First, the term árabi, establishes an evident link with the former dominant language. Secondly, the toponym Juba can be interpreted as an exclusive geographic label by outsider groups, and in particular by the Dinka political elite. In the following excerpt, the interviewee brings spontaneously out the question of the glottonym in relation with the non-recognition of Juba Arabic as a official language.

Excerpt 8. Speaker 7 11 jenub-īn / fi eyy maḥall / gi=wónusu árabi juba // lakín / I think / hukúma / I don't know // dayr-īn i-duss-u árabi / lakín nas lísa gi=wónusu árabi // wa nas ma gi=wónusu bes árabi sákit // law mašē-t fi šári / hum gi=wónusu árabi juba // ána šāyif al=muškila al=kabīr // hum ma ligó ísim le árabi de // šenú hum ma dayr-īn i-nad-ú árabi juba ↑ wa ma éndu ísim le úo // le lúga de // so / úmon bi=jérib ámulu ignore lúga de // lakín you can't ignore al hája ey zol gi=híbu de // árabi juba is growing everywhere // de reality lázim hukúma bitána árif // law hum ma dayr-īn i-samm-ú árabi juba / i-fattiš-u ísim táni / árabi jenúbi / wála hája // [...] law šuf-ta kiswahili / éndu a lot of Arabic words // lakín geyrú ísim to / it developed now / it is a new language / [...] so / the same thing / lázim i-fattiš-u ísim / yála árabi juba bi=kún kamán official language // "South Sudanese people, in every place, speak Juba Arabic. I think, the government, I don't know, want to hide Arabic, but people still speak Arabic, and they don't speak whatever kind of Arabic. If you go into the streets, they speak Juba Arabic. I think that the main problem is that they did not find a name for this Arabic. Why don't they want to call it Juba Arabic and they don't have a name for it, for this lúga? So, they just try to ignore this lúga, but you cannot ignore something that is appreciated by everyone. Juba Arabic is growing everywhere. This is a reality that our government should know. If they don't want to call it Juba Arabic, let us look for another name, like árabi jenúbi ("Southern Sudanese Arabic") or something else. If you look at Kiswahili, it has many Arabic words, but they changed its name, it has evolved and it became a new language. So, the same thing, let us look for another name so that Juba Arabic can eventually be a national language." This young returnee is a heritage speaker of Juba. He shows many instances of morphological integration from Sudanese Arabic and he switches frequently towards English. Despite his limited proficiency in Juba Arabic, the interviewee puts emphasis on the linguistic distinctiveness of the pidgincreole and calls for the official recognition of this lúga. In his view, the status of national language necessarily passes through the adoption of a new and more inclusive glottonym as, for example, árabi jenúbi "Southern Sudanese Arabic". Similar to speaker 6 in excerpt 6, the interviewee further strengthens his position with an explicit reference to Swahili, which is supposed to has evolved as an independent official language because the adoption of a new glottonym.

An opposite position concerning the glottonym árabi jûba is the one expressed in this last excerpt of interview. Excerpt 9. Speaker 5 Juba / was where the word separation was first pronounced // There was no alternative for the future of South Sudan / except to go in the independence // Juba here // So you could see / this was a starting point / for nationalism / and patriotism // and if Juba Arabic is called Juba Arabic / I would wonder it could offend any liberally minded person // Any person who is not ignorant / actually // As we can see, speaker 5, argues for an overt association between the toponym Juba and the rise of national South Sudanese identity. Consequently, he takes position against the adoption of a new glottonym for Juba Arabic. This means that the "indigenous" border constructed by the interviewee in excerpt 5 is not perceived as necessarily in contrast with the appreciation of the vehicular nature of Juba Arabic nor with the recognition of its local glottonym, which is considered symbolically associated with South Sudanese nationalism.

6.

Conclusions In this paper, I have tried to illustrate how the construction of linguistic borders in South Sudan interacts with locally available language ideologies. Evidence shows that the indexicalities of Juba Arabic, when compared to Sudanese Arabic, are interwoven with complex social discourses. First and foremost, in contrast to the State's decision to consider Juba Arabic as a variety of the former dominant language, speakers of the pidgincreole overtly construct linguistic borders between Juba Arabic and its lexifier.

The most significant border marker for Juba Arabic speakers who do not speak Sudanese Arabic is still represented by the low degree of mutual intelligibility between the two languages (excerpt 1). Juba Arabic-Sudanese Arabic bilingual speakers, on their part, make use of different features for constructing linguistic borders. The question could be then raised why certain linguistic features are only meaningful for some speakers and not for others. A reasonable answer is that metalinguistic representations of Juba Arabic are increasingly driven by the ideological hegemony enacted by the State through its discourse on "indigenous languages". In truth, metalinguistic representations focusing on linguistically relevant features like verbal morphosyntax (excerpt 2) are gradually giving way to more politically emblematic representations which emphasize symbolic linguistic features such as the presence of "indigenous" loanwords in Juba Arabic (excerpts 4 and 5). The tendency to the "indigenization" of language is also reflected in the politically emblematic use of verbal morphosyntax for the construction of community-internal borders between South Sudanese long-term residents and returnees from north Sudan (excerpt 3). It is also worth noting that speakers do not usually refer to phonological features for the construction of linguistic border between Juba Arabic and Sudanese Arabic. The erasure [START_REF] Irvine | Language ideology and linguistic differentiation[END_REF] of this relevant linguistic domain may be due to the fact that Juba Arabic shares some of its phonological features with other vehicular varieties of Arabic (cf. 3). Thus, speakers tend to eliminate details that are inconsistent with an linguistic identification of Juba Arabic.

As a further matter, the social construction of linguistic borders in South Sudan is evidently related to the emergence of a national identity. In contrast to the fact that Juba Arabic is not (and probably will never be) recognized as a national language, speakers consider it as the only interethnic means of communication that can guarantee a national unity against the rampant tribalism enacted by the State by means of its language policies (excerpts 6 and 7). This view, which is also reinforced by the gradual upscaling of Juba Arabic in the local system of language classification (excerpts 2, 6, 8), is not antithetical with the representation of the pidgincreole as an "indigenous language". This shows that the emerging national identity of South Sudan is inexorably linked with the notion of "indigenousness" inherited from colonial policies.

This paper inevitably simplifies a sociolinguistic reality that is much more complex. However, it reveals that linguistic borders constructed by speakers are not ideologically driven per se, since they can also be constrained by cognitively prominent features which are meaningful for descriptive linguists. Nevertheless, linguistic borders inevitably interact with local linguistic ideologies which move metalinguistic representations away from a factual linguistic reality. This state of affairs eventually brings to the fore the need for a more nuanced and multifaceted approach to language borders than those founded exclusively on ideological premises.

Bibliography

  Abdelhay, A. 2007. The Politics of Language Planning in the Sudan: the Case of the Navaisha Language Policy. Ph. D. dissertation, University of Edinburgh.

In this regard, it is interesting to note that, in the Arabic-based Ki-Nubi creole of Uganda and Kenya, the term rután simply means "language" (see Luffin

2003).

Male, 38 years old, born in Juba, secondary education, L1 Juba Arabic, L2 Sudanese Arabic, L3 Bari.

Male, 31 years old, born in Juba, higher education, L1 Juba Arabic, L2 Kuku, L3 English, moved toMaseru from 1990Maseru from until 2005. .

Male, 59 years old, born in Juba, secondary education, L1 Mundari, L2 Juba Arabic, L3, Sudanese Arabic, L4 English, moved toKampala from 1972Kampala from until 1979. .

Female, 25 years old, born in Juba, secondary education, L1 Juba Arabic, L2 Arabic, L3 Abukaya.

Male, 27 years old, born in Khartoum, secondary education, L1 Sudanese Arabic, L2 Juba Arabic, L3 English, L4 Swahili, moved to Nairobi from 1995 until 2011.