Larbi Rhazi
email: larbi.rhazi@unilasalle.fr

Lyes Lakahal

Ophelie Andrieux

Narcisse Niamba

Flore Depeint

Damien Guillemet

Relationship between the molecular characteristics of Acacia gum and its functional properties

Introduction:

Acacia gum, known as gum Arabic (GA), is a hydrocolloid complex, polysaccharide-based gum exudate. The gum is secreted by the trunk, branches or fruit of Acacia trees (i.e Acacia senegal and Acacia seyal) in response to a scission, injury or fungal infection. Acacia trees are natives to dry areas of the sub-saharian region, from Senegal to East Africa, of which Sudan, Chad, and Nigeria produce nearly 95% of GA exported to the world market.

The dried gummy exudate, consisting of soluble fibers of low viscosity, is an interesting edible ingredient for several industrial applications (Williams & Phillips, 2009). Thanks to its technological features, the GA is extensively used in the food, pharmaceutical, printing, textile, and cosmetic industries. Added to liquid food systems, they can adjust and improve their rheological properties. GA generally function as stabilizer, emulsifier, film former, thickener, flocculant and surface-finishing agent. These intrinsic properties make GA a recognized food additive (E414). They are found in the production of beverages, confectionery, emulsions, flavor encapsulations, bakery products and brewing [START_REF] Verbeken | Exudate gums: occurrence, production, and applications[END_REF].

These exceptional functional properties are thought to be tightly related to the composition and characteristics of molecular structure of GA. The molecular structure of GA consists of a mixture of highly branched and acidic polysaccharides. The main linear chain is composed of a core of 1,3-linked β-D-galactopyranose monomers with branches linked through C-6 but also C-4 or C-2. The branched chains consist of galactose and arabinose terminated by rhamnose and glucuronic acid (Al-Assaf et al., 2005a, 2005b;[START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in Acacia seyal in relation to Acacia senegal[END_REF]. Gel permeation and hydrophobic chromatography studies have confirmed that Acacia senegal gums are composed of three distinguish components. The main population (88-90%) consists of arabinogalactan-peptide (AG) displaying a molecular weight about 300 kDa. The second component accounts for ~10%, corresponds to arabinogalactan protein complex (AGP) with a 9% protein content and molecular weight starting from 1400 kDa. Finally, a much smaller population (~2%) consists of glycoprotein (GP) and displays the highest protein content (25-50%) [START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF]. These different values may change depending on several factor such as origin, age, and storage conditions.

The biochemical composition and molecular characteristics of GA can be affected by internal factors such as Acacia specie and age of trees. They can also change depending of external factors such as tree location, weather conditions, site and tapping method. The post-harvesting processes can also impact the molecular features and, as a result, the technological properties of GA (Al-Assaf et al., 2005a, 2005b;[START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Hassan | Studies on Acacia gums: Part III molecular weight characteristics of Acacia seyal var. seyal and Acacia seyal var fistula[END_REF][START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in Acacia seyal in relation to Acacia senegal[END_REF]. Very little data exists on the importance of the complex and versatile molecular variables contributing to the establishment of the functional properties of GA. Therefore, the overall objective of this work was to identify molecular variables that have the strongest effect on GA quality using partial least squares regression (PLS-R). PLS analysis is a useful statistical method that is suitable when there are more independent variables than response variables and there is multicollinearity among the independent variables [START_REF] Abdi | Partial least squares regression and projection on latent structure regression (PLS Regression)[END_REF].

One of the most popular variable selection methods in partial least squares (PLS) projection method is Variable Influence on Projection (VIP), also known as Variable Importance in the Projection scores [START_REF] Wold | PLS-partial least squares projections to latent structures, 3D QSAR in drug design[END_REF]. VIP scores are useful to give an estimate of the contribution of a given predictor to a PLS regression model.

In this work PLS-R analysis combined with VIP scores on molecular characterization data will be used to understand and gain more insight about the relationship between molecular variables of GA and their functional properties. The results from this investigation will help gain a better understanding of the qualitative and quantitative molecular mechanisms controlling the establishment of GA qualities.

Material and methods:

Acacia gum samples

Forty seven GA samples from Acacia senegal were provided by NEXIRA company (Rouen, France). Spray-dried industrial powder went through dissolution, filtration and pasteurization.

Grade quality of samples were sorted by their stabilizing potency for oil in water emulsion (turbiscan method). Here we propose to characterize and quantify functional potency of GA in emulsion with technical variables such as viscosity, emulsifying property EP (ability to decrease size distribution of oil droplet) and emulsion stability ES (ability to maintain size distribution over different conditions and time).

Viscosity measurement

For each GA sample, dry matter was quantified (105 °C, 5 h). GA samples were dispersed in water at 35% based on dry matter and left to hydrate (3 h, 20 °C). Then, viscosity determination was performed using Brookfield LVF viscometer (spindle 2, 60 rpm) (AMETEK Brookfield, Hadamar-Steinbach, Germany) with a spindle 2 and a rotational speed of 12 rpm.

Emulsion preparation

For each GA sample, continuous phase was prepared with a 20% GA solution (w/w) in water containing 0.75% of citric acid, with a settling period of hydration (1 h) after powder dispersion in liquid. Oil phase consisting of a a 50:50 (w/w) mix of limonene (three-fold distillation) and glycerol ester of wood rosin (Ester Gum 8BG) was used as weighting agent in order to minimize density difference between continuous and oil phase. 14.25% of oil phase was dispersed in GA solution during a shearing treatment performed using Ultra Turrax® T50 (maximum speed, 3 min) in order to obtain a coarse pre-emulsion. After a 10 min rest, solution went through 2 passes of homogenization including each 2 contiguous stages, respectively at 4060 and 580 psi.

Quantification of emulsifying property (EP) and emulsion stabilization (ES)

Emulsifying property was defined by the ability of GA to decrease the size distribution of the final emulsion. The EP variable was determined by d (0.9), the diameter of distribution for which 90% of oil droplet sizes were smaller. Individual EP was quantified on fresh homogenized emulsion by laser light scattering using Malvern Mastersizer Hydro2000 (Malvern Panalytical, Orsay, France).

Stabilizing property was defined by the ability of GA to maintain an emulsion stable (ie. minimized deviation of size distribution) throughout different stressing factors. The ES variable was calculated by the individual relative difference (expressed as percentage) of d (0.9) after a destabilizing stress applied on emulsion. Destabilizing stress corresponded to a heat treatment (90 °C, 20 min) followed by an accelerated stability study (50 °C, 3 months).

Size exclusion chromatography coupled with High Multi-Angle Laser Light

Scattering detection (SECMALLS) system and procedure GA samples (60 mg) were dispersed and incubated at room temperature for 48 h with 30 mL of 0.03 M sodium chloride containing 0.02% (w/v) azide, used as preservative. (Gelman Sciences, France) was installed between the main pump and the Eclipse system.

Sodium chloride (0.03 M) containing 0.02% (w/v) azide, was used as the mobile phase and filtered through a 0.1 mm membrane (Gelman Sciences, France). The flow rate of eluent was set at 0.5 mL.min -1 .

All necessary constants for molar mass (MM) distribution calculation were determined using a value of 0.141 mL.g -1 as refractive index. A pullulan110 standard was used to normalize the diodes of the MALLS detector. The calculated Mw of pullulan110, pullulan200 and pullulan800 standards were reasonably close to the nominal value.

The recorded MALLS, RI and UV data were processed using Astra software (v. 5.3.4.13,Wyatt Technology). The MM and radius were obtained by fitting the MALLS data using the Zimm method, performing a first order fit to the data collected at scattering detectors 5-17.

MM and radius can be obtained by MALLS combined with concentration measurements by applying the Rayleigh-Gans-Debye approximation (Equation 1),

𝐾 * 𝑐 𝑖 𝑅 𝜃 = 1 𝑀 𝑖 𝑃(𝜃) + 2𝐴 2 𝑐, ( 1
)
where Rθ is the excess Rayleigh scattering over and above that of the solvent due to a solute of concentration ci for slice i of the fractionated sample. K* is an instrumental constant dependent on the wavelength and refractive index increment (dn/dc) of the solute in the particular solvent.

Mi is the weight-average molar mass for the solute. P(θ) is the scattering function that describes the angular dependence of the scattered light intensity from which the mean-square radius <r2> is derived. A2 is the second virial coefficient and captures non-ideality due to solvent-solute interactions; it is expected to be vanishingly small in a dilute fractionated sample.

(𝑀𝑤 = Σ𝑐 𝑖 𝑀 𝑖 Σ𝑐 𝑖 ), (2)
(𝑀𝑛 = Σ𝑐 𝑖 Σ 𝑐 𝑖 𝑀 𝑖 ⁄ ), (3)
(𝑀𝑧 = Σ𝑐 𝑖 𝑀 𝑖 2 Σ𝑐 𝑖 𝑀 𝑖 ), (4)
(𝑅𝑤 = Σ(𝑐 𝑖 <𝑟2> 𝑖 ) Σ𝑐 𝑖 ), (5)
(𝑅𝑛 = Σ( 𝑐 𝑖 𝑀 𝑖 <𝑟2> 𝑖 ) Σ( 𝑐 𝑖 𝑀 𝑖 ) ), (6)
(𝑅𝑧 = Σ(𝑐 𝑖 𝑀 𝑖 <𝑟2> 𝑖 ) Σ(𝑐 𝑖 𝑀 𝑖 ) ), (7)
The average weight-average molar mass (Equation 2) is derived by measuring Rθ at multiple angles for each slice i of the fractionated sample. The concentration of each slice, ci, is measured by RI detector. The number-average molar mass is defined as Equation 3. The z-average molar mass is defined as Equation 4. The weight-average mean square radius is calculated as Equation 5. The number-average mean square radius is determined as Equation 5. The z-average root mean-square radius (Equation 7) is derived from the mean-square radii. Calculations of uncertainty-weighted average (Mavg) molar mass, polydispersity (Mw/Mn), uncertainty-weighted average (Ravg) mean square radius, RI mass fraction, relative proportion form UV and UV area/RI area ratio (UV/RI ratio) were calculated..

Partial least square regression

PLS-R analyses were performed on the collected data using the PLS-Toolbox 7.5 (Ei-genvector Research, Inc., Manson, WA, USA) in Matlab software version R2018a. All relevant parameters will be developed in the results section.

Results and discussion:

Acacia gum functional attributes

The emulsifying activities of gum samples were determined by characterizing emulsions using particle size analysis with laser light scattering . This technique is more suitable for that kind of measurement than the turbidometric technique [START_REF] Buffo | Factors affecting the emulsifying and rheological properties of gum acacia in beverage emulsions[END_REF]. The EP expressed as d(0.9) ranged from 1.21 to 1.70 with an average value of 1.41 and coefficient of variation (CV) of 8.53% (

Functional properties correlation

Linear correlation coefficient (r²) was calculated between paired functionality properties. The negative correlation between EP and viscosity was very weak (r² = 0.16). There was no correlation between ES and viscosity (r² = 0.01). The negative correlation between ES and EP was very weak (r² = 0.18). Those data confirm a high level of independency between the three functional properties and that we should consider and assess them independently.

The lack of correlation between functional properties agrees with industrial observations where the potency of EP is not systematically associated with ES efficacy over time. Stoke's law describes the velocity of emulsion sedimentation or creaming, based on a formula significantly and positively affected by viscosity [START_REF] Chanamai | Creaming Stability of Flocculated Monodisperse Oil-in-Water Emulsions[END_REF]. Creaming instability process of emulsion, however, is a different and later event than deviation of oil droplet (ES), the latter reflecting coalescence (merging of oil droplets). We should therefore assume that viscosity could help maintain and stabilize large oil droplets, but is not implicated in the preliminary coalescence process.

SEC-HPLC analysis of Acacia gum

Figure 1 shows a typical set of chromatograms representing UV detection (214 nm), which is mostly sensitive to the peptide bond, refractive index (RI) response, light scattering (LS) response and molar mass distribution (MMD) versus elution time using NaCL (2 g.L -1 ) as mobile phase. Chromatograms are comparable to previously published SEC-HPLC GA chromatograms [START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF][START_REF] Padala | Adsorption of Gum Arabic, Egg White Protein, and Their Mixtures at the Oil-Water Interface in Limonene Oil-in-Water Emulsions[END_REF][START_REF] Atgié | Gum Arabic in solution: Composition and multi-scale structures[END_REF]. However, our separation displays higher resolution in comparison with those obtained previously [START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF]. Integration of RI peak areas enabled determination of the recovery of injected mass and calculation of the percentage of GA components. In this investigation, the elution recovery was 100%. For all gum samples, the RI profile showed two distinct peaks. The first small peak (elution 37-43 min, max 42 min) was attributed to the arabinogalactan-protein (AGP) population as reported previously [START_REF] Randall | Fractionation and characterization of gum from Acacia senegal[END_REF]. The second large peak (elution 43-55 min, max 47 min) was attributed to the arabinogalactan (AG) fraction. The UV elution profiles were significantly different from the RI profiles showing a huge peak with shoulder, covering the first two fractions mentioned earlier, and a resolved peak (50 min), proteinaceous in nature and identified as the glycoprotein (GP) population [START_REF] Randall | Fractionation and characterization of gum from Acacia senegal[END_REF]. The LS and RI profiles were not comparable and did not superimpose. The LS profiles clearly displayed two response peaks (41 and 45 min), allowing MM and size determination of the AGP and AG fractions, respectively.

A Debye Plot (Zimm fit) of Rθ/K*c vs. sin2(θ/2) for the GA components enabled a good estimate of the molecular features. According to MMD (Figure 1), AGP and AG population displayed a broad range of MM ranging from about 1.4×10 6 to over 10×10 6 g.mol -1 and from about 1.8×10 5 to 1.4×10 6 g.mol -1 , respectively. In addition, the average mean square radius values for AGP and AG ranged from 30 to over 100 nm and from 20 to 30 nm, respectively.

Acacia gum molecular characteristics

Molecular parameters were determined for each population (Figure 1): AGP population (36-43 min), AG population (43-48.8 min), GP population (48.8-52 min) and whole GA (36-52 min).

Most sample molecular parameters displayed a CV > 5% showing great dispersion in our sample population (Table 2, 3 and4). Table 2. Molecular characteristics of molar mass and mass fraction of polymeric components obtained from SE-HPLC-UV-MALLS-RI, for 47 samples of GA. The Mass fraction % determined using RI signal particularly showed GP content values significantly different from those found in the literature. They were high compared to the most referenced data [START_REF] Randall | Fractionation and characterization of gum from Acacia senegal[END_REF][START_REF] Islam | A review of recent developments on the regulatory, structural and functional aspects of gum arabic[END_REF][START_REF] Renard | Acacia senegal Gum: Continuum of Molecular Species Differing by Their Protein to Sugar Ratio, Molecular Weight, and Charges[END_REF][START_REF] Al-Assaf | Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM TM ): Part 1-Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier[END_REF]; while they are lower than that recently announced by Atgié et al where the authors found that this population represents 27 wt % of gum Arabic mass [START_REF] Atgié | Gum Arabic in solution: Composition and multi-scale structures[END_REF]. Mostly, differences were due to differences in matrix of fractionation columns. Indeed, plausible explanation is the co-elution of GA and GP. The more important the co-elution is, the more the GP content will be overestimated. This is due to the resolution power of the columns used.

Based on preliminary studies (not shown) Superose 6 Increase 10/300 GL (GE Healthcare Europe GmbH, Velizy-Villacoublay, France) and TSK-Gel G4000 SW (TOSOH Bioscience, Japan) columns led to GP content mean equivalent to 5% while serial shodex columns (OHPAK SB 804HD and 806HD columns) led to values ranging from 9 to 16% depending on the samples analyzed (raw gum, atomized gum). The advantage of the later columns was the best resolution of AGP that why they have been chosen for the current study.

The average Mw for the AGP population of samples ranged from 3.33×10 6 to 6.02×10 6 g.mol -1 with an average value of 4.33×10 6 g.mol -1 and CV of 16.26% while that for the AG population oscillated between 5.80×10 5 to 7.12×10 5 g.mol -1 with an average value of 6.41×10 5 g.mol -1 and CV of 4.48% (Table 2). In addition, the average Mw ranged from 4.08×10 5 to 6.09×10 5 g.mol -1 for whole GA. Comparing CV, the average Mw variability was more important for AGP fraction.

The values obtained agree reasonably with those cited in the literature. However, values of Mw calculated for our samples were higher than those determined previously [START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF][START_REF] Flindt | Studies on acacia exudate gums. Part V. Structural features of Acacia seyal[END_REF][START_REF] Siddig | Studies on acacia exudate gums, part IV. Distribution of molecular components in Acacia seyal in relation to Acacia senegal[END_REF][START_REF] Renard | Structure of arabinogalactan-protein from Acacia gum: From porous ellipsoids to supramolecular architectures[END_REF][START_REF] Gashua | Characterisation and molecular association of Nigerian and Sudanese Acacia gum exudates[END_REF]. This could be due to differences in column, solubilization, elution conditions and or sample nature. The mean of AGP dispersity (Mw/Mn) was 1.39 suggesting that this population is constituted of heterogeneous polymers. The associated CV was about 8.06%, suggesting variation among samples. Lower dispersity and CV were noted for AG and GP fractions (Table 2). The measured average size parameters showed a strong variation. The Rw of AGP showed the lowest CV while Ravg of GP had the highest CV (47.27%). The mean z-average size (Rz) of AGP, AG and GP were 56.62, 18.05 and 23.44 nm, respectively. Recently, these fractions were found to have diameters of 20 and 10 nm, for AG and GP, respectively [START_REF] Gashua | Characterisation and molecular association of Nigerian and Sudanese Acacia gum exudates[END_REF] while consensus was reached for AGP with maximum dimension of 64 nm [START_REF] Renard | Structure of arabinogalactan-protein from Acacia gum: From porous ellipsoids to supramolecular architectures[END_REF][START_REF] Gashua | Characterisation and molecular association of Nigerian and Sudanese Acacia gum exudates[END_REF].

PLS-R VIP

Collinearity tests were carried out. These tests showed that our independent parameters all without exception showed high Variance Inflation Factor (VIF) values indicating the presence of multicollinearity. Thus, PLS-R analysis was used.

Using PLS-R, VIP was useful in understanding and selecting the most influential and important explicative variables for each technological property. In literature, VIP scores provide a useful measure to select variables that best explain variance [START_REF] Farrés | Chemometric evaluation of Saccharomyces cerevisiae metabolic profiles using LC-MS[END_REF][START_REF] Ceglar | Impact of meteorological drivers on regional inter-annual crop yield variability in France[END_REF][START_REF] Mkhabela | Characterising the most critical climatic parameters that impact the quality of spring-wheat (Triticum aestivum L.) on the Canadian Prairies using partial least squares (PLS) analysis[END_REF].

Normally, the average of the squared values of the VIPs is equal to 1. The criterion of VIP value with greater than 1 is typically set as a cutoff point describing the relative contribution of the predictor variables to the PLS latent variables [START_REF] Mehmood | A review of variable selection methods in Partial Least Squares Regression[END_REF]. However, there are usually different types of data structures. Thus, the cut-off point has to be determined according to the type of data structure. The VIP threshold can then vary between 0.8 and 1.2. Determining the appropriate cut-off point is not straightforward. If the cutoff value is too high, some crucial variables will be missing. Conversely, too low a cut-off value will result in the absence of more unrelated variables. In this study, the cutoff threshold of VIP was estimated at 0.8 based on the use of detection outlier with boxplot obtained from added noise variables according to the methodology proposed by Akarachantachote et al [START_REF] Akarachantachote | CUTOFF THRESHOLD OF VARIABLE IMPORTANCE IN PROJECTION FOR VARIABLE SELECTION[END_REF]. PLS analyses showed that there were many independent variables with a VIP score ≥0.8 and most of them exhibited a VIP score ≥1 (Table 5). Therefore explicative variables with VIP scores between 0.8 and 1.2 were considered as the very important variables while those having VIP scores ≥1.2 were designated as the most critical factors [START_REF] Mkhabela | Characterising the most critical climatic parameters that impact the quality of spring-wheat (Triticum aestivum L.) on the Canadian Prairies using partial least squares (PLS) analysis[END_REF].

PLS-R VIP and viscosity

The best PLS model accounted for 92% of the variability in viscosity. This confirms that all main molecular variables implicated in viscosity have successfully been considered.

Nine variables contributed significantly to the variability of viscosity, as shown by the VIP score ≥0.8, while four variables were identified as the most critical factors (VIP score ≥1.2) that contributed to viscosity variability (Table 5). Based on VIP scores, the most critical factor was Mz of AGP, followed by Mz of whole GA polymers, Mw and Mn of AGP with VIP score of 3.72, 3.65, 2.92 and 2.54, respectively. Results suggested that the most influential parameters were tightly related to the MM of GA polymers but also in high extent to AGP. Furthermore, the average gyration radius (Ravg) of AGP population exhibiting a VIP score of 0.95 contributed to viscosity variability. Although, mass fraction percentage of AGP is considered to impact the steady viscosity and moduli of whole gum dispersion [START_REF] Al-Assaf | Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM TM ): Part 1-Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier[END_REF], our data (VIP of 0.29) did not show importance of the relative percentage of AGP population on viscosity variability. Usually, the relationship between viscosity and percentage of AGP have been only determined by correlation studies. All investigations including ours have shown positive correlations. In our case, this parameter exhibited a significant correlation with the viscosity (R 2 =0.63).

However, these correlations seems to be due to co-linearity with other explicative parameters since in our study and that of Al-Assaf et al [START_REF] Al-Assaf | Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM TM ): Part 1-Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier[END_REF], the percentage of AGP also correlated to many other parameters such as the MM and size of AGP that were positively correlated to viscosity. This demonstrated that correlation studies are not always relevant for determining the roles that explicative parameters play in establishing technological properties. It is very likely that the conclusions obtained are due to the change associated with the increase in the AGP percentage, mainly the increase in their molar mass and size. This is in accordance with the VIP scores of theses parameters. This is the first time where a clear relationship have been established between AGP MM distribution and viscosity. Our findings are coherent with previous studies where the intrinsic viscosity was found to be tightly related to the gum MM and the hydrodynamic volume that control the hydration status and conformation of macromolecules [START_REF] Robinson | Viscosity-molecular weight relationships, intrinsic chain flexibility, and dynamic solution properties of guar galactomannan[END_REF][START_REF] Mudgil | X-ray diffraction, IR spectroscopy and thermal characterization of partially hydrolyzed guar gum[END_REF][START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF]. Furthermore, authors reported an increase of 20-fold in viscosity with a rise of MM from 4.24×10 5 to 14.5×10 5 g.mol -1 during maturation of GA [START_REF] Al-Assaf | Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM TM ): Part 1-Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier[END_REF]. The role of the AGP MM could be explained by the fact that polymers have a tendency to aggregate in aqueous solution via intermolecular hydrophobic interactions between the polypeptide component and/or hydrogen bonds between the polysaccharide residues [START_REF] Mahendran | New Insights into the Structural Characteristics of the Arabinogalactan-Protein (AGP) Fraction of Gum Arabic[END_REF][START_REF] Renard | Structure of arabinogalactan-protein from Acacia gum: From porous ellipsoids to supramolecular architectures[END_REF]. Hence, AGP constitute an elementary molecular background for aggregate establishment that affect the viscosity which is in accordance with previous report (Li et al., 2009). They found that the viscosity diminishes as function of molecules disaggregation.

PLS-R VIP and emulsifying property

The best PLS model explained 76% of the variability in EP. This indicates that a majority of molecular variables have been taken into account for EP assessment. There are still other molecular parameters implicated, not studied here, which could complete this relationship.

29 variables from the 46 studied contributed significantly to the variability of the EP, as shown by the VIP score ≥0.8, while 13 variables were classified as the most critical factors (VIP score ≥1.2) that contributed to the EP variability (Table 5). In comparison, the number of variables contributing to this rheological parameter was much higher than that of variables controlling the viscosity. The implication of molecular structure of GA in EP is more complex than for viscosity and involved a wide range of variables. Almost all the most critical factors were related to MM variables including MM of AGP, whole gum and GP. With VIP score of 2.16 and 2.14, Mn and Mavg of AGP were first and second among the most critical factors. It was clear that AGP MM were the most contributing factors in the establishment of the emulsifying activity. Gyration radius parameters with VIP score ≥0.8 were also crucial for this qualitative property mainly Ravg of GP which was determined as one of the most critical factors with VIP value of 1.64. In addition Rn, Rw and Rz of GP exhibiting VIP scores ≥0.8 outlined that not only MM but also the size of GP were essential determinants of EP. Similarly Ravg of AGP and whole gum contributed to the EP variability as they showed VIP scores ≥0.8. Once more, AGP% did not have a high VIP score meaning that relative percentage of AGP did not influence the formation of emulsion while mass fraction percentage of AG, with VIP value of 0.83, was shown to be essential for this qualitative parameter.

Interestingly, variables related to protein content (UV signal) were shown to be partially involved in the variability of EP. In addition, among these variables only UV area/RI area of GP population were identified as the most critical factors with VIP score of 1.35. UV% of GP displaying a VIP value of 0.84, showed a relevant role of this population in the EP variability. VIP values of AGP UV/RI ratio and AGP UV% and UV% of AG revealed that these factors which are related to peptides contributed to emulsification behavior.

PLS-R VIP and emulsion stabilization:

The best PLS model justified 77% of the variability in the ES percentages. Similarly to EP, this indicates that a majority of molecular variables have been taken into account for ES assessment and that still other molecular parameters could be partially implicated. 27 variables were found significantly responsible for the variability of the ES parameter, as displayed by the VIP score ≥0.8, while 15 variables were determined as the most critical factors (VIP score ≥1.2) that contributed to the ES variability (Table 5). In comparison with the viscosity, the number of the very important variables explaining the variability of the ES were three time larger, showing a much more complex relationship. Despite the absence of correlation between emulsification parameters (EP, ES) and viscosity described earlier, similar results to viscosity were noted. All MM parameters of different GA fractions were shown to contribute to ES. More interesting, MM of GP was among the most critical factors, and exhibited the highest values of VIP. The impact and role of population size was quite similar in variable number comparing to EP. However for ES, only Ravg of AGP was determined as the most critical factor while Ravg of GP, AG and whole gum as well as Rz of AGP and whole gum were considered as very important variables. As for EP, the AGP mass fraction did not show an important role. But, the excellent stability of emulsions observed for GA with high content of AGP seemed to due to the fact that the higher content of AGP would confers a thicker interfacial layer and provides more effective steric stabilizing effect, due to the larger hydrodynamic size of the AGP fraction (Dickinson et al., 1991;[START_REF] Dickinson | Hydrocolloids at interfaces and the influence on the properties of dispersed systems[END_REF][START_REF] Han | Effect of arabinogalactan protein complex content on emulsification performance of gum arabic[END_REF]. As for EP, mass fraction percentage of AG was revealed to influence and contribute to ES as its VIP score was 0.84.

In addition, it was determined that the peptides represented by UV% of AGP, UV/RI ratio of AGP and UV/RI ratio of GP were involved in ES. UV% of AGP were one of the most critical factors while UV/RI ratio of AGP and UV/RI of AG were among the most important factor. 22 molecular parameters were common for the explanation of EP and ES. Without exception, all MM variables of all GA fractions were shown to be implied in emulsification variables and most of them were so as the most critical factors. The MM of AGP was closely involved in EP variability while MM of GP contributed strongly to ES variability. EP was also shown to be dependent of gyration radius of GP and relative protein content. Thus, our study suggested that the MM of GA polymers depended on their heterogeneous composition of proteins and polysaccharides. In addition, it was obvious that the MM controlled gyration radius and surface active of polymers. It suggested that the quality of GA which is a well-established natural emulsifier agent, depends on polymer MM and that importance resides in their ability of self-interaction due to their composition of amphiphilic proteins and hydrophilic polysaccharides. Therefore, our study complements previous reports showing the impact of some intrinsic parameters on GA properties. It has been reported that the relative proportion of the three main fractions governs the emulsification properties [START_REF] Dickinson | Surface activity and emulsifying behaviour of some Acacia gums[END_REF]. Further, thanks to polypeptide chain hydrophobicity and carbohydrate hydrophilicity, AGP population showed to be the main responsible of the emulsifying behavior and interfacial properties of the GA [START_REF] Dickinson | Surface activity and emulsifying behaviour of some Acacia gums[END_REF][START_REF] Randall | Fractionation and characterization of gum from Acacia senegal[END_REF][START_REF] Fauconnier | Characterization of Two Acacia Gums and Their Fractions Using a Langmuir Film Balance[END_REF]. complex molecular structure implication than viscosity.. Population percentage (mass fraction) of AGP and GP were not implicated in any functional properties while for AG there was a slight implication in only EP and ES. Molar mass (MM) of AGP and GP however played a critical role in all functional properties but with a higher implication in viscosity compared to EP and ES. ES and EP were also impacted by MM of AG. Some other specificities between functional properties have been showed, as radius gyration of GP was more implicated in EP compared to radius gyration of AGP which was more implicated in ES. We should also highlight the weight of protein content (mainly from AGP and GP) for both EP and ES, while it is not implicated in viscosity. These data have shown the importance of understanding the role of component that have abilities to self-association via different kinds of surface interactions as has been reported in literature.

With this study, this is the first time that statistical tests have been performed on 47 samples of regular Acacia senegal in order to correlate and rank molecular characteristics in accordance with functional properties. As perspectives, the same approach will be performed to deepen our understanding of the Acacia gum technological properties by including other physicochemical parameters such as zeta potential, solution permittivity, potentiometric titration.

Figure 1 .

 1 Figure 1. Size exclusion chromatogram of Acacia senegal gum sample using UV-MALLS-RI

  Table1). CV is a dimensionless measure of the dispersion of our samples, indicating that our samples were moderately dispersed. The ES percentages oscillated between 3.49 and 69.90 with an average of 18.38. As judged by the very high CV (75%), gum samples presented a larger range of dispersion. The third studied parameter was viscosity. Samples

	exhibited an average viscosity of 847.89 with CV of 13.85% demonstrating medium to high
	dispersion.
	Parameters	Viscosity mPa.s	Emulsifying Property (EP) d (0.9) µm	Emulsification Stabilisation (ES) Δd (0.9) %
	minimum a	666	1.21	3.49
	maximum a	1134	1.70	69.90
	Average b	847.89	1.41	18.37
	SD	117.46	0.12	13.80
	CV %	13.85	8.52	75.14

a Mean value of three experiments. b Mean value for 47 samples

Table 1 .

 1 Descriptive variables of functional properties Viscosity, EP and ES for 47 samples of Acacia gum.

Table 3 .

 3 Characteristics of molecular size distribution of polymeric components obtained from SE-HPLC-UV-MALLS-RI, for 47 samples of GA.

	Parameters	Rw (nm)	Rz (nm)	Ravg (nm)
	AGP
	minimum a	34.60	46.30	35.70
	maximum a	48.10	72.30	59.10
	Average b	39.72	56.62	44.50
	SD	3.35	6.21	8.38
	CV %	8.44	10.98	18.84

AG

b Mean value of 47 samples of GA

Table 4 .

 4 Protein moiety proportions and proteins to polysaccharides ratio obtained from SE-HPLC-UV-RI, for 47 samples of GA.

			AGP	AG		GP		Whole gum
	Parameters UV/RI UV (%)	UV/RI UV (%)	UV/RI UV (%)	UV/RI
	minimum a 264.00	24.19	64.53	48.70	39.42	7.17	80.82
	maximum a 381.86	55.58	80.28	63.73	89.35	13.32	114.34
	Average b	309.42	35.68	69.91	54.32	63.44	10.65	94.82
	SD	26.73	7.92	3.15	3.20	8.83	1.12	8.45
	CV %	8.64	22.21	4.51	5.90	13.93	10.56	8.91
			a Mean value of three experiments each
				b Mean value of 47 samples of GA

Table 5 .

 5 Variable Importance in the Projection scores (VIP) for viscosity, emulsifying property (EP) and emulsification stabilization (ES).

	Variables		VIP
		Viscosity	Emulsifying property (EP)	Emulsification stabilization (ES)
	AGP
	Mn	2.54	2.16	1.75
	Mw	2.92	1.82	1.30
	Mz	3.71	1.25	1.23
	Mavg	1.05	2.13	1.22
	Mw/Mn	0.02	0.01	0.01
	Rn	0.23	0.46	0.41
	Rw	0.25	0.87	0.56
	Rz	0.60	0.74	1.01
	Ravr	0.95	1.09	1.35
	Mass fraction RI	0.29	0.32	0.33
	UV Area/RI Area	0.69	1.15	1.13
	UV Area %	0.49	1.10	1.32
	AG
	Mn	0.70	0.93	0.93
	Mw	0.91	0.77	0.81
	Mz	0.63	1.01	1.09
	Mavg	0.55	1.21	1.33
	Mw/Mn	0.00	0.01	0.00
	Rn	0.28	0.77	0.78
	Rw	0.32	0.38	0.77
	Rz	0.29	0.35	0.72
	Ravr	0.23	0.58	0.82
	Mass fraction RI	0.34	0.83	0.84

Acknowledgement

The authors are pleased to acknowledge the technical assistance of Lucie Lequeux.

More recently, it has been proposed that the aggregation of polyproline helices in GA, through CH...π interactions, would play a major role in the emulsification mechanism of GA [START_REF] Sethuraman | Is Gum Arabic a Good Emulsifier Due to CH...π Interactions? How Urea Effectively Destabilizes the Hydrophobic CH...π Interactions in the Proteins of Gum Arabic than Amides and GuHCl?[END_REF]. Our results which highlighted the weight of MM, relative proportion of proteins and protein to polysaccharide ratio (UV/RI ratio) in the establishment of GA properties, could explain previous statements describing the polymer aggregation [START_REF] Al-Assaf | Molecular associations in acacia gums[END_REF][START_REF] Li | Feature Selection for Partial Least Square Based Dimension Reduction[END_REF][START_REF] Renard | Structure of arabinogalactan-protein from Acacia gum: From porous ellipsoids to supramolecular architectures[END_REF][START_REF] Renard | Structure of glycoproteins from Acacia gum: An assembly of ring-like glycoproteins modules[END_REF][START_REF] Apolinar-Valiente | Recovery, structure and physicochemical properties of an aggregate-rich fraction from Acacia senegal gum[END_REF].

Considering these reports and our data, it is obvious that polymer MM governed by polysaccharides and polypeptides composition explains GA rheological behaviors that would be linked to the capacity of polymers to self-aggregate. The aggregation is due to molecular association via proteinaceous hydrophobicity interactions between aminoacid side chains in the polypeptide component and/or intermolecular hydrogen bonds between the polysaccharide residues. Then, the amount and the characteristics of the protein fractions could be considered crucial for intermolecular association. Further completion of PLS model for variability explanation could provide more information. In this aim, we should consider studies performing similar statistic tests and including other molecular variables related to protein composition as aromatic aminoacids, known for their higher hydrophobic behaviors (ie. UV 280/214 ratio; [START_REF] Bérépion | Incidence de la composition chimique et des propriétés physico-chimiques des gommes en acacia sur leurs propriétés en émulsion[END_REF]) or markers related charge as Zeta potential for each emulsion performed with fractionated AGP, AG and GP [START_REF] Jayme | Zeta potentials of gum arabic stabilised oil in water emulsions[END_REF].

Conclusions:

This study confirmed that the three functional properties, viscosity, emulsifying property (EP) and emulsion stabilization (ES) were rather independent and pointed the relevance to evaluate them independently. Molecular variables from SE-HPLC-MALLS considered in this study were relevant to assess relationship with viscosity, EP and ES as reflected by PLS model implication in their variability (92, 76 and 77% respectively). EP and ES have a much more


Marina Réquilé

Dubàn Gonzàlez Alvarez

Stéphane Delanaud

Larbi Rhazi

Véronique Bach

Flore Depeint

Hafida Khorsi-Cauet

Use of a combination of in vitro models to investigate the impact of chlorpyrifos and inulin on the intestinal microbiota and the permeability of the intestinal mucosa

Keywords: Caco-2/TC7, chlorpyrifos, inulin, dysbiosis, microbiota, SHIME®

come

Use of a combination of in vitro models to investigate the impact of chlorpyrifos and inulin on the intestinal microbiota and the permeability of the intestinal mucosa

Marina Réquilé, Dubàn O Gonzàlez Alvarez, Stéphane Delanaud, Larbi Rhazi, Véronique Bach, Flore Depeint, Hafida Khorsi-

1) Introduction

In 2016, a total of 59,300 metric tons of pesticides were used in Francemaking the country the third largest user of pesticides worldwide and the prime user in Europe. Some pesticides are considered to be endocrine disrupters; hence, day-to-day exposure is likely to have serious, irreversible effects on organ maturation and development [START_REF] Lassiter | Exposure of neonatal rats to parathion elicits sex-selective reprogramming of metabolism and alters the response to a high-fat diet in adulthood[END_REF][START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]. Furthermore, some pesticides can cross the placental barrier rapidly, and may therefore affect foetal development. The organophosphorothionate pesticide chlorpyrifos (CPF) has been extensively used in agriculture as a means of controlling insect pests, and is considered to be the most commonly used pesticide worldwide [START_REF] Harishankar | Efficiency of the intestinal bacteria in the degradation of the toxic pesticide, chlorpyrifos[END_REF].

Exposure to CPF during early foetal development is known to perturb the gut's environment, permeability and functions [START_REF] Condette | Chlorpyrifos Exposure During Perinatal Period Affects Intestinal Microbiota Associated With Delay of Maturation of Digestive Tract in Rats[END_REF]Reygner et al. 2016b). A pesticide's impact on the digestive tract is of particular interest, since this is the first organ to come into contact with ingested food contaminants. However, there are few data on CPF's mechanisms of action in the digestive tract.

The intestinal microbiota acts as an interface between foodstuff and digestive cells and thus has a role in the intestine's maturation and in digestion. Furthermore, the intestinal microbiota enables the assimilation of nutrients, helps to digest fibre, participates in the synthesis of certain vitamins, and regulates the absorption of fatty acids, calcium and magnesium. It also stimulates immune defences, and acts as a protective barrier against pathogenic microbes and toxins. The microbiota's composition and diversity change in successive stages from birth onwards, when the neonate passes from the in utero compartment (often considered to be sterile) into a microorganism-rich environment. By the age of 3 years, the microbiota's composition and diversity have stabilized and thereafter remain relatively stable throughout life -although some changes have been observed in older adults [START_REF] Mariat | The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age[END_REF]). The microbial environment can nevertheless be disturbed from time to time by factors such as antibiotic treatments, immune system failure, infections, changes in dietary habits, and other changes to the environment. This disturbance is often characterized by a decrease in the numbers of beneficial microorganisms and a simultaneous increase in the numbers of potentially pathogenic microorganisms [START_REF] Kwok | Characterization of fecal microbiota across seven Chinese ethnic groups by quantitative polymerase chain reaction[END_REF]. Like any ecosystem, the intestinal microbiota is usually able to return to its initial state a few weeks after a disturbance; this phenomenon is referred to as resilience (Antonopoulos et al. 2009). In contrast, persistent instability or imbalance (i.e. intestinal dysbiosis) has often been linked to pathologies such as inflammatory bowel disease [START_REF] Sokol | Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients[END_REF], obesity, and diabetes [START_REF] Zhao | The gut microbiota and obesity: from correlation to causality[END_REF].

Bacterial translocation corresponds to the passage of viable bacteria from the gastrointestinal flora through the intestinal mucosa's barrier (the lamina propria) to the mesenteric ganglia and then to normally sterile internal organs (such as the spleen and the liver) [START_REF] Steffen | Relationship between cecal population levels of indigenous bacteria and translocation to the mesenteric lymph nodes[END_REF]. Translocation may result from functional alterations in the mucosa and/or increased microbial loads in the intestine.

We have previously reported that chronic, low-dose exposure to CPF causes intestinal dysbiosis in an in vitro model of the digestive tract (the Simulator of the Human Intestinal Microbial Ecosystem, SHIME®), with an increase in the numbers of some bacterial strains and a corresponding decrease in others [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]Reygner et al. 2016a). However, the SHIME® does not take account of a substance's impact on epithelial cells. Using cultures of Caco-2 cells, Tirelli et al. showed that exposure to 250 µM CPF was associated with tight junction perturbations, the loss of barrier effects, and increased epithelial permeability [START_REF] Tirelli | Effects of the pesticide clorpyrifos on an in vitro model of intestinal barrier[END_REF]). However, this Caco-2 cell-based model does not take account of the preliminary metabolism of CPF in the lumen of the digestive tract. Little information is available on CPF-degrading bacteria in the intestinal tract. Some recent studies have evaluated CPF's effects in pure cultures. Treatment with CPF (200 mg/L) was associated with an increase in the counts of four different lactic acid bacteria, which suggests that these strains can tolerate high concentrations of CPF [START_REF] Cho | Biodegradation of Chlorpyrifos by Lactic Acid Bacteria during Kimchi Fermentation[END_REF]. Other studies showed that Streptomyces spp. and Pseudomonas spp. strains were able to tolerate CPF in a mixture of pesticides [START_REF] Singh P B | Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos[END_REF][START_REF] Fuentes | Enhanced Removal of a Pesticides Mixture by Single Cultures and Consortia of Free and Immobilized Streptomyces Strains[END_REF]. Lastly, another study found that a number of intestinal bacteria had CPF-degrading properties and generated strain-specific metabolites, such as CPFoxon (also an endogenous metabolite), trichloropyrinidol and diethylphosphate [START_REF] Harishankar | Efficiency of the intestinal bacteria in the degradation of the toxic pesticide, chlorpyrifos[END_REF].

Although these studies have mechanistic relevance, they involved isolated bacterial cultures. As such, they are not really comparable with our use of a complex ex vivo microbiota matrix.

Although a few dynamic, all-in-one in vitro models of the intestinal tract have been developed, they are complex from a technical standpoint [START_REF] Marzorati | Studying the host-microbiota interaction in the human gastrointestinal tract: basic concepts and in vitro approaches[END_REF]. The choice of model also depends on the hypothesis to be tested [START_REF] Von Martels | The role of gut microbiota in health and disease: In vitro modeling of host-microbe interactions at the aerobe-anaerobe interphase of the human gut[END_REF]. Here, we decided on the sequential application of the SHIME® and Caco2/TC7 cell cultures. A two-model combination is becoming the gold standard for in vitro investigations. We focused on the impacts of the luminal environment and the treatment-modified colonic microbiota on a variety of mucosal parameters, including the tight junction regulation, mucosal permeability to macromolecules, bacterial translocation, and the regulation of epithelial functions.

As mentioned above, a large body of data shows that colonic dysbiosis can be directly treated via dietary intervention. The administration of prebiotics (e.g. dietary fibres such as inulin) might constitute a means of reducing CPF's harmful effects. The impact of inulin on human health has been extensively studied [START_REF] Roberfroid | Prebiotic effects: metabolic and health benefits[END_REF]Reygner et al. 2016b). Hence, we decided to determine whether or not CPF's effects on selective bacterial growth and bacterial metabolite levels could be reversed by introducing the prebiotic inulin into the diet.

Prebiotics are non-digestible food ingredients that benefit the host by acting as a substrate for certain bacteria in the digestive tract. Interactions and competition between beneficial bacteria and potentially pathogenic bacteria can positively or negatively influence the microbiota. Indeed, the growth of beneficial bacteria (Bifidobacterium spp. and Lactobacillus spp.) can prevent potentially pathogenic bacteria from colonizing the digestive tract by modifying the pH or stimulating the production and release of short-chain fatty acids (SCFAs) [START_REF] Roberfroid | Prebiotic effects: metabolic and health benefits[END_REF].

The objective of the present study was to better understand CPF's impact on the intestinal luminal ecosystem and the epithelium in vitro. We therefore combined two systems, one mimicking the physical-chemical conditions in the lumen and the other mimicking a working epithelial mucosa. The impact on the lumen was assessed by measuring selected bacterial populations and bacterial metabolites, whereas the impact on the epithelium was assessed by measuring the expression of tight junction proteins. The study secondary objective was to investigate the putative protective effect of a prebiotic (inulin) on these parameters.

2) Materials and Methods

The overall experimental design is summarized in Figure 1.

Chemicals

Chlorpyrifos (purity: 99.8%) was purchased from LGC Standards (Molsheim, France). The stock solution (10 mg/mL) was prepared in vegetable oil. Next, 3.5 mL of the stock solution were mixed with 96.5 mL of the primary feed (Molly et al. 1993b), giving a 0.35 mg/mL working solution (stored at 4°C). 50 g of inulin (kindly provided by Cosucra, Warcoing, Belgium) were added to 1 L of primary feed, mixed for 30 min, and stored at 4 °C.

The SHIME® model

The SHIME® is a dynamic in vitro model of the human microbial ecosystem (Molly et al. 1993b), and has been described in detail by Joly et al [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]. Six double-jacketed reactors are connected in series via peristaltic pumps. The reactors respectively mimic the stomach, two segments of the small intestine (the duodenum/jejunum and the ileum/cecum), and the ascending, transverse, and descending segments of the colon. Each of the colon segments was connected to a pH probe, which recorded the change of pH in the medium. Twice a day, the pH was adjusted to the physiological value or range (Table 1). The residence time in each reactor is determined by its working volume, and the total transit time is 86 hours. The compositions of the SHIME®'s feed and pancreatic juice have been described elsewhere (Molly et al. 1993a;[START_REF] Boever | Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder[END_REF]. To mimic feeding patterns, 200 mL of primary feed were added to reactor 1 every 8 hours.

Treatment

The colon reactors were inoculated with a mixture of faecal microbiota from five healthy adult volunteers (including males and females), none of whom had suffered from gastrointestinal diseases or taken antibiotics in the previous six months [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]. In view of the system's technical parameters, no more than two conditions can be tested in parallel. To ensure the absence of selective degradation of the microbiota during storage, samples were used fresh for each experiment. However, samples from the same set of volunteers were used to minimize the variability of the microbial profiles. The in vitro environment had stabilized after 14 days, as proven by stable pH fluctuations within the physiological range (Table 1) and a stable redox potential. Day 0 (D0) was defined as the untreated control value. Samples were collected from the different reactors on three non-consecutive days, to increase the number of replicates. Immediately after collection of the final untreated samples, 10 mL of the CPF solution (3.5 mg) and 200 mL of primary feed containing the inulin (10 g) were injected daily into reactor 1 for 30 days. Only the first of the day's three meals contained the treatments. Fifteen and 30 days after the initiation of treatment (i.e. on D15 and D30), samples were collected from reactors 4 to 6 (representing the ascending, transverse and descending colon, respectively) and used immediately for microbiological studies or stored at -20 °C prior to analysis. Again, with a view to increasing the number of replicates, triplicate samples were collected on non-consecutive days. We also performed CPF-only and inulin-only SHIME® treatments.

Microbiological evaluation

Serial dilutions (1/10) of fresh samples from reactors 4 to 6 were prepared in Ringer solution (Oxoid, Basingstoke, England). The microorganisms were identified using standard microbiological techniques and various nonselective, selective, semi-selective and indicator bacterial growth media [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]. All anaerobic plates were incubated in a Bactron anaerobic chamber (Sheldon Manufacturing, Inc., Cornelius, OR, USA) for 5 days.

Isolated colonies grown on Petri dishes were counted using an automatic colony counter (Scan® 500, Interscience, St Nom la Bretèche, France) and expressed in log(CFU)/mL. Although bacterial cultures detect (by definition) only culturable, viable strains, these are the ones of interest; our ultimate objective is to investigate bacterial translocation through the epithelial layer and to the basal compartment.

Short-chain fatty acids

Fermentation slurries were centrifuged at 14,000 g for 10 min, and the supernatants were filter-sterilized (VWR International, Strasbourg, France). 100 μL of internal standard and 50 μL of sulfuric acid were added to 900 μL of supernatant. The SCFA content was determined as described elsewhere [START_REF] Lecerf | Xylo-oligosaccharide (XOS) in combination with inulin modulates both the intestinal environment and immune status in healthy subjects, while XOS alone only shows prebiotic properties[END_REF]) using a Focus GC gas chromatograph (Thermo Scientific, Illkirch, France) equipped with a flame ionization detector and a gas chromatography column (length: 30 m; inner diameter: 25 mm; film: 1 μm), with H2 as the carrier gas at a flow rate of 1 mL/min. The different SCFAs were identified according to the elution peaks' respective retention times, and then quantified by comparison with standard curves.

Caco-2/TC7 cell culture

The Caco-2/TC7 intestinal cell line was kindly provided by Dr M. Rousset (INSERM U178, Villejuif, France).

The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum, 1% non-essential amino acids (100 X) and 100 units/mL penicillin-streptomycin. All these reagents were purchased from Gibco (Fisher Scientific SAS, Illkirch, France). Isolated cells were seeded directly onto polycarbonate inserts in tissue-culture-treated 6-well plates (diameter: 24 mm; pore size: 1 μm; Fisher Scientific SAS) at a density of 250,000 cells/insert. The medium was refreshed every other day for 21 days; at this time point, the epithelial cells had differentiated fully and formed a tight, functional monolayer (as assessed by the transepithelial electrical resistance [TEER]).

Treatment

The treatment was initiated at 21-23 days post-seeding. Frozen samples from the SHIME® experiment were thawed and either used as such or as the supernatant (SHIME® luminal medium) after centrifugation at 5000 rpm for 5 min and filtration through a 0.2 µm pore membrane). Each of the triplicate samples collected from reactor 4 at D0, D15 and D30 was added instead of culture medium to a separate well (n=3). We also included samples from CPF-only and inulin only SHIME® treatments in this experiment. Cells were exposed to the various treatments for 6 or 24 hours.

Epithelial layer integrity

The integrity of a monolayer of differentiated Caco-2/TC7 cells growing on inserts was monitored by measuring the TEER [START_REF] Tirelli | Effects of the pesticide clorpyrifos on an in vitro model of intestinal barrier[END_REF]). After a 15 min stabilization period, the TEER was recorded at room temperature using chop-stick electrodes (Millicell®ERS-2, Millipore, Molsheim, France) and expressed in Ω x cm², according to the following equation: TEER = (Ω cell monolayer -Ω cell-free filter) x filter surface area

Genetic expression

All reagents and working kits were purchased from Qiagen SA (Courtaboeuf, France). Caco 2/TC7 cells were collected in RNAprotect Cell Reagent prior to storage. After centrifugation (14,000 g, 1 min), total RNA was extracted from the cell pellet using an RNeasy® Mini Kit, according to the manufacturer's instructions. The mRNA content in the flow-through was determined spectrophotometrically using a NanoDrop 2000 system (Thermo Scientific, Illkirch, France). Complementary DNA (cDNA) was generated by reverse transcription of 200 ng mRNA using a QuantiTect® Reverse Transcription kit, according to the manufacturer's instructions. The real-time quantitative polymerase chain reaction (RT-PCR) was performed on 5 μL of cDNA (diluted 1/10 in RNasefree water), 1 μL of primers (Primerdesign Ltd, Southampton, UK), 4 μL of RNase-free water and 10 μL of QuantiFast® SYBR® Green PCR Master Mix in a total of 20μL per sample (in triplicate). The target genes and corresponding primers are listed in Supplementary Table 1. Following activation of the HotStart Taq DNA polymerase (95 °C, 5 min), 40 cycles of amplification (denaturation at 95 °C, 10 sec followed by annealing/extension at 60 °C, 30 sec) were performed with fluorescence detection at the end of each extension step. This was followed by a melting curve analysis (from 60 °C to 95 °C) for quality control of the amplicons.

Relative expression was calculated with regard to untreated cells, and both GAPDH and ATP5B were used as reference genes.

Secretion of an inflammatory marker

The secretion of interleukin-8 (IL-8) into the apical medium was studied by assaying 50 μL samples with a Bio-Plex Pro TM Human Cytokine IL-8 Assay Kit (Bio-Rad Laboratories Inc., Marnes-la-Coquette, France), according to the manufacturer's instructions. The plate was read using flow cytometry (MACS QUANT® Analyser, Miltenyi biotech, Bergisch Gladbach, Germany). The IL-8 was then quantified by reference to a standard curve.

Statistical analyses

Data were expressed as the mean ± standard deviation (SD). The various experimental conditions were compared in a one-way analysis of variance. All statistical analyses were performed using StatView software (version 5.0, Abacus Concepts Inc., Berkeley, CA, USA). The threshold for statistical significance was set to p <0.05.

3) Results and Discussion

Impact of CPF and inulin co-treatment on the bacterial intestinal environment, as assessed with the SHIME® model

The data presented here relate to reactor 4, since (i) the ascending colon is where most of the inulin is metabolized in vivo, and (ii) the intestinal cells were treated with samples taken from this reactor (in vivo, the ascending colon is the closest part of the colon to the small intestine's mucosa). In contrast to the marked dysbiosis observed previously at a dose of 1 mg/d in vitro [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]Reygner et al. 2016a) or at the equivalent dose of 3.5 mg/d in vivo (Reygner et al. 2016b), exposure of the SHIME® to the dose of 3.5 mg/d used here significantly reduced the Lactobacillus and the Bifidobacterium counts (Figure 2C and 2D), while counts of other selected bacteria (including pathobionts like Enterobacteriacea and Enterococcus) were not significantly modified (figure 2A and2B). Inulin increased the Lactobacillus and Bifidobacterium counts but also reduced the Enterococcus count and the Enterobacteriacea counts; however, none of these changes were statistically significant. The combination of CPF and inulin significantly reduced the Enterococcus count, while the Bifidobacterium and Lactobacillus counts were not significantly affected. The Enterobacteriacea count decreased over time but the change was not statistically significant. The overall effect (as summarised in Figure 2) partially prevented an increase in the pathobionts count and was associated with a non-significant decrease in the numbers of lactic acid bacteria.

Molecular profiling of the whole microbiota would probably provide more accurate information on the combined effects of CPF and inulin, since some of the affected bacteria might not have been cultured in our tests.

There are only limited data on the interactions between CPF and the human microbiota. However, in vivo and in vitro experiments [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF][START_REF] Condette | Chlorpyrifos Exposure During Perinatal Period Affects Intestinal Microbiota Associated With Delay of Maturation of Digestive Tract in Rats[END_REF]Reygner et al. 2016b, a) demonstrated that a 30-day period of CPF treatment was associated with increase in the numbers of potentially pathogenic microbiota and a decrease in the numbers of beneficial microbiota in all segments of the colon. The literature data indicate that CPF exposure in the rat is associated with higher Staphylococcus spp. counts in all segments of the digestive tract [START_REF] Condette | Chlorpyrifos Exposure During Perinatal Period Affects Intestinal Microbiota Associated With Delay of Maturation of Digestive Tract in Rats[END_REF]. In contrast to the negative impact of CPF, van de Wiele et al. (van de [START_REF] De Wiele | Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem[END_REF][START_REF] Van De Wiele | Inulin-type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects[END_REF]) and others (Allsopp et al. 2013;[START_REF] Sivieri | Prebiotic effect of fructooligosaccharide in the simulator of the human intestinal microbial ecosystem (SHIME® model)[END_REF]) have confirmed the prebiotic effects of chicory inulin on Bifidobacterium and Lactobacillus in all segments of the colon. To the best of our knowledge, there are no previous reports of CPF and inulin co-treatment in the SHIME® or in similar models; hence, we can only refer to our own data to discuss inulin's role with respect to CPF. The data from our previous experiments in the rat at the same CPF concentration (3.5 mg/day) indicate that in vitro and in vivo co-treatments with CPF and inulin have similar effects on bacterial populations (Reygner et al. 2016b).

Total SCFA levels increased under all three treatment conditions, although the increase was only statistically significant with the CPF+inulin mixture; this was probably due to the small number of replicates in each condition (Figure 3). However, the profiles of individual SCFAs differed as a function of the treatment. The presence of CPF alone increased the acetic acid content (from 18.49 ± 2.91 µmol/mL at D0 to 25.07 ± 7.68 µmol/mL at D30), the presence of inulin increased the propionic and butyric acid levels (from 0.98 ± 0.60 µmol/mL at D15 to 3.81 ± 0.39 µmol/mL at D30 for proprionate, and from 0.62 ± 0.40 µmol/mL at D15 to 0.91 ± 0.66 µmol/mL at D30 for butyrate), and the CPF+inulin mixture increased the propionic acid level (from 7.57 ± 0.60 µmol/mL at D0 to 15.36 ± 2.88 µmol/mL at D30). It is generally considered that the ratios between the three main SCFAs (Figure 3) are more meaningful than the absolute concentrations. These values gave a slightly different picture. CPF alone did not change the ratios -suggesting that all the bacteria responsible for saccharolytic hydrolysis are metabolically modulated by the pesticide in a similar manner. In the prebiotic group, we did not observe the shift in propionate and butyrate production (an increase in total, propionic and butyric acids in all segments of the colon) observed in a number of in vitro SHIME® experiments (van de [START_REF] De Wiele | Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem[END_REF]) following inulin treatment for 3 weeks. We even observed an unexpected fall in propionate's contribution at D15, although we could not link it to specific bacterial modulations. The CPF+inulin mixture was associated with a large, statistically significant decrease in the acetic acid ratio in R4 and a significant increase in the propionic acid ratio. The butyric acid ratio was not affected (Figure 3C). Overall, increased SCFA synthesis creates an acidic environment in the colon; in vivo, this protects against pathogens such as Enterococcus and the Enterobacteriaceae, as shown by our microbial measurements in the CPF+inulin condition. The pH was measured daily as part of the system check; a decrease in pH was observed as early as day 7 (data not shown). However, the system set-up requires the pH to be maintained within a predefined range, and so the pH was adjusted twice a day -preventing further acidification. Our observation of elevated SCFA release constitutes additional evidence of inulin's ability to relieve (at least in part) the damage caused by exposure to CPF. The increase in SCFA levels may appear to be inconsistent with the transient increase in numbers of Staphylococcus measured at D15 (data not shown) time but is in line with the inhibition of Enterococcus as early as D15. Further investigations at earlier time points might provide a better understanding of the relationship (if any) between bacterial species and saccharolytic metabolites. Furthermore, SCFAs constitute an important energy source for colonocytes, and also influence colonic function by stimulating sodium and water absorption and modulating motility [START_REF] Topping | Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides[END_REF]. This is important when considering the impact of SHIME® extracts on intestinal cells and the latter's function.

Impact of CPF and inulin on Caco-2/TC7 cells following digestion in the SHIME®.

In the CPF-only, inulin-only and CPF+inulin experiments in the SHIME®, samples from reactor 4 were tested on Caco-2/TC7 cells.

Exposure to SHIME® extracts led to a fall in the TEER over time. The baseline (D0) TEER values were above that of the control (culture medium alone) for the CPF treatment and the inulin treatments but not for the CPFinulin mixture. Although this was probably due to the more complex molecular composition of the medium, it is not unclear why the three conditions were not equivalent at baseline. Compared with the corresponding D0 control, total (unfiltered) SHIME® extracts led to a loss of tissue integrity after as little as 2 hours, and so were not used further. Although filtered SHIME® extracts also induced a drop in the TEER (Supplementary Table 2), the TEER value did not differ significantly from the control value (D0, untreated SHIME® medium) for up to 8 hours. The time-dependent drop in TEER suggests that the permeability of the Caco-2/TC7 monolayer increases quickly following exposure to SHIME® extracts. Observation of the wells showed that at 24 hours, the cell layer had detached from the membrane surface. Following further tests, we determined that a 1:5 dilution of the SHIME® sample did not affect the stability of the epithelial layer, as measured by the TEER and dextran-FITC diffusion to the basal compartment (data not shown). However, the data presented here come from the exposure of undiluted SHIME® extracts. Given that the TEER dropped quickly, we examined the functional status of treated cells at 6 hours. We are aware that these data may not be fully representative of physiological exposure.

The D0 samples of cells exposed to SHIME® samples showed no variations in the expression levels of structural genes associated with cell differentiation and epithelial function (ACTB, VIL1, ALPI, SI) (Table 2), suggesting that the epithelium remained functional under these reference conditions. At D15, cells exposed to CPF-treated extracts had significantly lower expression levels of all tested genes (overall decrease: 30%), with the exception of the differentiation genes ALPI and SI (no variation). Inulin-treated extracts did not change overall expression levels, even though ALPI was increased at D15, whereas cells exposed to the CPF-inulin SHIME® samples showed a large, time-dependant and significant increase in expression of the structural gene VIL1 (2-fold) and of the tight junction genes CDH1 (2-fold), OCLN (4-fold) and TJP1 (5-fold), as well as differentiation genes ALPI and SI relative to SHIME® samples collected on D0 (controls). In summary, we found that CPF alone tended to inhibit the expression of tight junction and structural genes, whereas inulin and CPF/inulin co-treatment tended to increase expression. Taken as a whole, these observations suggest that the pesticide disrupted the tight junctions, while the mixture strengthened the tight junction activity. With regard to genes coding for bacterial and luminal receptors, the expression level of TLR4 rose strongly and significantly at D30 (8-fold) with CPF+inulin but not with CPF alone or inulin alone, suggesting that a specific luminal environment was created by changes in bacterial populations other than those measured here.

To the best of our knowledge, similar in vitro experiments have not been reported in the literature. Hence, we can only relate the present in vitro data to our previous in vivo findings. The increase in TJP1 and OCLN expression might compensate for CPF-induced leakage through the intermembrane space observed in vivo [START_REF] Condette | Chlorpyrifos Exposure During Perinatal Period Affects Intestinal Microbiota Associated With Delay of Maturation of Digestive Tract in Rats[END_REF]. The strong increase in TJP1 gene expression may be a sign of increased production of the encoded tight junction protein zonula occludens-1 [START_REF] González-Mariscal | Tight junctions and the regulation of gene expression[END_REF]. We found that samples exposed to CPF alone for 30 days were associated with downregulated expression of the CDH1 gene (coding for the transmembrane glycoprotein E-cadherin). Several studies have shown that low CDH1 expression may be a risk factor for gastric cancer (Al-Moundhri et al. 2010). E-cadherin has an important role not only in regulating the morphogenesis of normal and neoplastic tissues but also in tumour invasion and metastasis [START_REF] Gao | Meta-analysis: tumour invasion-related genetic polymorphisms and gastric cancer susceptibility[END_REF]. With regard to genes encoding functional proteins, we observed that the expression levels of SI and ALPI were increased by CPF alone and CPF+inulin but not by inulin alone. Overexpression of ALPI may be associated with a recovery from an initial toxic episode because alkaline phosphatase is known to be involved in reducing inflammatory responses [START_REF] Lallès | Intestinal alkaline phosphatase: novel functions and protective effects[END_REF]; this might be related to the early (D15) increase in the numbers of Staphylococcus. Furthermore, the enzyme has been linked to the expression of Toll-like receptor (TLR) 4 and the inhibition of IL-8 secretion (Chen and Ouyang 2010). However, recent studies have shown that contact between inulin and gut epithelia cells can modify epithelial tight junction integrity and signals related to the immune response [START_REF] Vogt | Immunological properties of inulin-type fructans[END_REF]. Lastly, Toll-like receptors are involved in bacterial sensing, and have an important role in controlling the homeostasis of the intestinal epithelium. Changes in TLR gene expression may be indicative of an imbalance in the microbiota and disturbance of the intestinal immune system [START_REF] Rakoff-Nahoum | Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis[END_REF]). The increase in TLR gene expression after exposure to CPF+inulin co-treatment may be related to the modulation of bacterial loads in general and that of the Enterobacteriaceae in particular [START_REF] Lu | LPS/TLR4 signal transduction pathway[END_REF]. It is not clear whether gene expression levels and bacterial counts were in our experimental setting. Changes in bacterial populations other than those measured here might have modulated gene expression in the Caco-2/TC7 cells.

The release of IL-8 increased in a time-dependant manner in the CPF-only condition (Figure 4; p <0.05 vs. D0) but remained low and stable for the inulin-only treatment and CPF-inulin conditions. These observations suggest that a proinflammatory signal is triggered by the pesticide but completely inhibited by the prebiotic.

These data are consistent with (i) our experiments on CPF-treated caco-2/TC7 cells (supplementary table 3), in which CPF induced IL8 secretion in a dose-dependent manner, (ii) modulation of the bacterial environment by the pesticide and the prebiotic (as measured in the SHIME® reactors), and (iii) the known anti-inflammatory role of probiotic bacteria, including Bifidobacterium strains (Park [START_REF] Ms | Assessment of lipopolysaccharide-binding activity of Bifidobacterium and its relationship with cell surface hydrophobicity, autoaggregation, and inhibition of interleukin-8 production[END_REF]Candela et al. 2008;[START_REF] Wang | Secreted factors from Bifidobacterium animalis subsp. lactis inhibit NF-κB-mediated interleukin-8 gene expression in Caco-2 cells[END_REF].

Based on CPF's molecular weight and its dilution in the SHIME® solutions, we estimate that the concentration of CPF in the colonic reactors is between 10 and 50 µM. At these concentrations, CPF in culture medium did not induce IL8 release into the luminal compartment. We thus hypothesize that CPF itself and CPF-induced factors in the SHIME® content may have synergistically induced an inflammatory response. Co-treatment with inulin and CPF had a marked impact by preventing the CPF-induced decrease in Bifidobacterium spp and modulating SCFA production in the colon reactors. As mentioned above, there are very few reports on CPF's effects on bacterial populations in vivo or in vitro [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF][START_REF] Condette | Chlorpyrifos Exposure During Perinatal Period Affects Intestinal Microbiota Associated With Delay of Maturation of Digestive Tract in Rats[END_REF]Reygner et al. 2016b, a). Most of the previous work used a CPF dose of 1 mg/d, which resulted in more severe dysbiosis (as measured by larger log differences at the end of the treatment period) than the higher concentration of 3.5 mg/d studied here. We suggest that some of the potentially discriminant bacterial responses to CPF observed at the lower concentration may have been saturated at the higher concentration -leading to changes that are independent of the bacterial species measured herein. Nevertheless, we observed a clear association between (i) luminal variables in the SHIME® and (ii) epithelial function, as measured in Caco-2/TC7 cell layers.

The relevance of combined in vitro models

Several studies have shown that the SHIME® is a relevant model for evaluating compounds such as probiotics/prebiotics [START_REF] Terpend | Arabinogalactan and fructo-oligosaccharides have a different fermentation profile in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME ®)[END_REF][START_REF] Chaikham | Effects of encapsulated Lactobacillus acidophilus along with pasteurized longan juice on the colon microbiota residing in a dynamic simulator of the human intestinal microbial ecosystem[END_REF], drug candidates [START_REF] Sousa | The gastrointestinal microbiota as a site for the biotransformation of drugs[END_REF], plant extracts [START_REF] Muñoz-González | Ability of human oral microbiota to produce wine odorant aglycones from odourless grape glycosidic aroma precursors[END_REF], and toxics [START_REF] Joly | Impact of chronic exposure to low doses of chlorpyrifos on the intestinal microbiota in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and in the rat[END_REF]. However, this model also presents certain limitations, such as the absence of (i) bacterial adhesion to the gut epithelium, (ii) microbiota-host crosstalk, and (iii) immune feedback and regulation [START_REF] Mainville | A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics[END_REF]. Some of these limitations can be circumvented by performing additional studies with Caco-2/TC7 cells. Under specific conditions, Caco-2/TC7 cells can form a simple, functional intestinal epithelium in which polarized cells have tight junctions and a brush border on the apical side. Furthermore, the Caco-2/TC7 monolayer presents a high TEER and can produce cytokines (such as IL-6, IL-8, TNFα, and IL-15) [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][START_REF] Verhoeckx | Food processing and allergenicity[END_REF]. Moreover, the Caco-2/TC7 monolayer's permeability coefficients in vitro are strongly correlated with absorption data measured in vivo in humans [START_REF] Verhoeckx | Food processing and allergenicity[END_REF].

By combining the SHIME® and Caco-2/TC7 models, it is possible to gain a better understanding of how different food components alter (and are altered by) the gastrointestinal environment and microbial populations before they reach the intestinal mucosa. The SHIME®-Caco-2/TC7 combination is not the first two-model system to have been reported. However, previously described combined models were used to investigate prebiotic activity with regard to mucosal health rather than a prebiotic's ability to protect the mucosa against an environmental disruptor.

In the present and previous studies, we found that CPF induces dysbiosis and a metabolic imbalance in the intestinal environment. While further analyses are required to fully characterize CPF's impact, it appears that one mechanism involves the inhibition of the growth and metabolism of lactic acid bacteria. These harmful effects were prevented (at least in part) by co-treatment with inulin, thanks to the recovery of health-promoting strains and the production of saccharolytic metabolites. We were not able to draw firm conclusions about barrier function parameters. It is nevertheless clear that CPF affected the activity of the mucosal barrier and the release of the chemokine IL-8, which might potentiate inflammation processes through the recruitment of immune effectors.

Lastly, CPF/inulin co-treatment was associated with an increase in TLR gene expression. Notably, this increase in expression was concomitant with the bacterial changes measured in the corresponding SHIME® samples.

However, prolonged apical exposure to SHIME® extracts (even in the absence of CPF) appeared to disrupt the Caco-2/TC7 monolayer. It might be of value to adjust the experimental set-up by (i) diluting the SHIME® extract in culture medium, and (ii) determining the optimal time range for measuring effects on gene expression and physiological parameters. Lastly, with a view to modelling the complexity of mucosal tissue more accurately, the combination of the SHIME® model and the Caco-2/TC7 model could be developed further by the inclusion of mucus-producing or immune cells. Even in its current state, the combination of these two in vitro models seems to be a relevant, ethically acceptable alternative to animal experiments.

Abbreviations

4) References

Allsopp P, Possemiers S, Campbell D, et al (2013)

  Abbreviations: CPF, chlorpyriphos; SHIME®, Simulator of the Human Intestinal Microbial Ecosystem; cDNA: complementary DNA; D, day; TLR, Toll-like receptor; TEER, transepithelial electrical resistance.

  Figure captions.

Figure 1 :

 1 Figure 1: Schematic representation of the experimental set-up and design.

Figure 2 :

 2 Figure 2: Changes over time in bacterial populations in reactor 4, following co-treatment with CPF and inulin.

Figure 3 :

 3 Figure 3: Short-chain fatty acid production in reactor 4 following treatment with CPF and/or inulin.

Figure 4 :

 4 Figure 4: IL-8 production by Caco-2/TC7 cells following exposure to SHIME® samples.

Figure

  Figure 4:

  Figure 4:

  An exploratory study into the putative prebiotic activity of fructans isolated from Agave angustifolia and the associated anticancer activity.

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	polymorphisms and gastric cancer risk. World J Gastroenterol 16:3432-3436
	Antonopoulos DA, Huse SM, Morrison HG, et al (2009) Reproducible community dynamics of the gastrointestinal
	microbiota following antibiotic perturbation. Infect Immun 77:2367-2375. doi: 10.1128/IAI.01520-08
	Candela M, Perna F, Carnevali P, et al (2008) Interaction of probiotic Lactobacillus and Bifidobacterium strains
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	modulation	of	IL-8	production.	Int	J	Food	Microbiol	125:286-292.	doi:
	10.1016/j.ijfoodmicro.2008.04.012

The authors thank the French Ministry of Research and Higher Education for funding the present study through MR's postgraduate fellowship. They thank the Picardy Regional Council for providing funding, and also thank David Fraser (Biotech Communication SARL, Ploudalmézeau, France) for copy-editing assistance. The authors also wish to thank Cosucra for kindly providing the inulin used in this project.

Tables

 Table 1:pH values, working volumes, and residence times for the SHIME® reactors corresponding to each segment of the human intestinal tract.

Reactor