Mohammed Iddir

Celal Degerli

Giulia Dingeo

Charles Desmarchelier

Thomas Schleeh

Patrick Borel

Yvan Larondelle

Torsten Bohn
email: torsten.bohn@gmx.ch

Whey protein

Keywords: Proteins, carotenoids, micellization, emulsion, digestion, lipid droplets, peristalsis

come

Introduction

Carotenoids and their metabolites are recognized as playing a role in the prevention of human diseases. Based on epidemiological studies, a positive association has been suggested between dietary intake and tissue concentrations of carotenoids and lowered risk of several chronic diseases [START_REF] Elliott | Mechanisms of genomic and non-genomic actions of carotenoids[END_REF], such as cardiovascular diseases and type 2 diabetes [START_REF] Sluijs | Dietary intake of carotenoids and risk of type 2 diabetes[END_REF], cancer [START_REF] Sharoni | Carotenoids and apocarotenoids in cellular signaling related to cancer: A review[END_REF], as well as reduced risk of age-related macular degeneration [START_REF] Arunkumar | What do we know about the macular pigment in AMD: the past, the present, and the future[END_REF]. These correlations may be attributed to their antioxidant and anti-inflammatory activity [START_REF] Krinsky | Carotenoids as antioxidants[END_REF], as well as their influence on the immune system [START_REF] Rao | Carotenoids and human health[END_REF], both possibly related to a number of transcription factors and nuclear receptors such as NF-κB [START_REF] Kaulmann | Carotenoids, inflammation, and oxidative stress--implications of cellular signaling pathways and relation to chronic disease prevention[END_REF] and RAR/RXR (Caris-Veyrat, Garcia, Reynaud, Lucas, Aydemir, & Rühl, 2017). In addition to these health relevant aspects, certain carotenoids, including β-carotene, constitute vitamin A active compounds, following their cleavage by β-carotene oxygenase 1 (BCO-1) in vivo and metabolism into retinal.

Before being absorbed, carotenoids must be released from the food matrix and dissolved in lipid droplets. Then, under the influence of digestive enzymes (especially lipase), pH, gastric peristalsis, and bile, carotenoids are incorporated into mixed micelles, allowing their uptake by the mucosa of the small intestine [START_REF] Bohn | Host-related factors explaining interindividual variability of carotenoid bioavailability and tissue concentrations in humans[END_REF]. Due to their lipophilic nature, the bioavailability of carotenoids, including β-carotene, is relatively low, typically between 10 and 20% [START_REF] Bohn | Bioavailabilty of non-provitamin A carotenoids[END_REF]Desmarchelier & Borel, 2017). Consequently, several studies have been investigating potential dietary as well as host-related factors influencing carotenoid bioavailability aspects, including their bioaccessibility and absorptive processes.

Regarding matrix-related factors, several studies have concluded that dietary lipids can significantly enhance carotenoid absorption, fostering micellization [START_REF] Bohn | Bioavailabilty of non-provitamin A carotenoids[END_REF][START_REF] Huo | Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids[END_REF]. Also, it has been reported that dietary fiber can decrease the Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 4 micellization of carotenoids, possibly by limiting enzymatic access and activity (O'Connell, Ryan, O'Sullivan, [START_REF] O'connell | Carotenoid micellarization varies greatly between individual and mixed vegetables with or without the addition of fat or fiber[END_REF]. Recently, divalent minerals at higher concentrations were suggested to compromise carotenoid bioaccessibility, likely by precipitating fatty acids and bile salts [START_REF] Biehler | Divalent minerals decrease micellarization and uptake of carotenoids and digestion products into Caco-2 cells[END_REF][START_REF] Borel | Dietary calcium impairs tomato lycopene bioavailability in healthy humans[END_REF]. In addition to these dietary factors, it was shown that host-related factors can modulate carotenoid bioavailability (reviewed in [START_REF] Bohn | Host-related factors explaining interindividual variability of carotenoid bioavailability and tissue concentrations in humans[END_REF], including conditions that alter the gastro-intestinal (GI) surface area or digestive enzyme secretion. For example, bariatric surgery has been associated with several physiological modifications, among them, gastric peristalsis movement, at least in part explaining the decrease of plasma carotenoid concentrations [START_REF] Granado-Lorencio | Depletion of serum carotenoid and other fat-soluble vitamin concentrations following obesity surgery[END_REF]. In another study, it was reported that Crohn's disease resulted in carotenoid malabsorption [START_REF] Edes | Essential fatty acid sufficiency does not preclude fat-soluble-vitamin deficiency in short-bowel syndrome[END_REF]. Other anomalies such as cholecystectomy (reducing the available amount of bile), and pancreatitis (leading to secretory insufficiency of digestive enzymes) have been proposed to compromise carotenoid bioavailability (Desmarchelier & Borel, 2017).

One factor that may impinge on carotenoid absorption, but has not yet received much attention, is the presence of proteins. For example, caseins have been successfully employed for micro-and nano-encapsulation of β-carotene, significantly improving its bioaccessibility [START_REF] Soukoulis | A comprehensive overview on the micro-and nano-technological encapsulation advances for enhancing the chemical stability and bioavailability of carotenoids[END_REF][START_REF] Yi | The physicochemical stability and in vitro bioaccessibility of beta-carotene in oil-in-water sodium caseinate emulsions[END_REF]. It was proclaimed that proteins may aid in the bioaccessibility of liposoluble dietary constituents in several ways. Following their adsorption to lipid droplet surfaces, proteins may stabilize oil-in-water (o/w) emulsions in the GI tract, attributed to the fact that proteins can be highly surface-active molecules [START_REF] Mun | Fluorescence imaging of spatial location of lipids and proteins during digestion of protein-stabilized oil-in-water emulsions: A simulated gastrointestinal tract study[END_REF][START_REF] Soukoulis | A comprehensive overview on the micro-and nano-technological encapsulation advances for enhancing the chemical stability and bioavailability of carotenoids[END_REF], and the formed particles tend to be highly negatively charged, preventing the aggregation of lipid droplets [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF].

However, the same study also showed that gliadin decreased the enzymatic degradation of lipids, likely by hindering digestive enzymes to adsorb to the droplet surfaces or binding to enzymes Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 5 directly, also implying potential negative influences of proteins on the micellization process. Whey protein isolate (WPI) in contrast was effective at inhibiting lipid oxidation of o/w emulsions [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF], which may contribute to limit oxidative degradation of sensitive molecules, as were caseino-phosphopeptides, which were proposed to chelate iron which may trigger oxidative degradation [START_REF] Kim | Antioxidant capacity of caseinophosphopeptides prepared from sodium caseinate using Alcalase[END_REF]. Furthermore, WPI also facilitated the formation of smaller lipid droplets; Salvia-Trujillo and co-workers showed that reducing the diameter of these particles was related to increased β-carotene bioaccessibility [START_REF] Salvia-Trujillo | Influence of particle size on lipid digestion and beta-carotene bioaccessibility in emulsions and nanoemulsions[END_REF].

It is assumed that the emulsifying properties are greatly influenced by the composition and structure of proteins. For instance, both hydrophobic and hydrophilic groups are abundant in WPI, highlighting its amphiphilic behavior [START_REF] Sharif | Current progress in the utilization of native and modified legume proteins as emulsifiers and encapsulants -A review[END_REF]. The structure of WPI is comprised of two main fractions, i.e. α-lactalbumin (123 amino acids) and β-lactoglobulin (162 amino acids), together accounting for approx. 75% of the whey proteins, and their conformation (predominantly α-helix and β-pleated sheet, respectively) affect their properties at the oil/water interface [START_REF] Chatterton | Bioactivity of β-lactoglobulin and αlactalbumin-Technological implications for processing[END_REF].

In the current study, we aimed to investigate the effect of a commonly consumed protein source, WPI, on the bioaccessibility of pure β-carotene, under a range of digestive conditions, as proteins were hypothesized to rather enhance bioaccessibility under marginal, i.e. somewhat insufficient digestive conditions. For this purpose, we employed a previously established in vitro GI consensus model [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF], and WPI was added at different concentrations, reflecting various fractions of the recommended dietary allowance (RDA) of proteins, i.e. 0, 25 and 50%. Micellization of co-digested β-carotene thus measured also under insufficient digestion parameters (i.e. shortened gastric digestion duration, limited concentrations of digestive enzymes, gastric peristalsis, bile, and a surplus of dietary lipids), such as possible in subjects with GI disorders.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003

6

Materials and Methods

Enzymes and chemicals

Pepsin from porcine gastric mucosa (powder, ≥250 U/mg, Art. No. P7000), pancreatin from porcine pancreas (activity equivalent to 4x USP specifications. Art. No. P1750), porcine bile extract (Art. No. B8631), as well as β-carotene standard (≥97% UV, >95% all-trans form according to supplier and own HPLC analysis, Art. No. 22040) were purchased from Sigma-Aldrich (Overijse, Belgium). Whey protein isolate (WPI) was obtained from Pure Nutrition USA (95% purity, California, USA) while the peanut oil, typically free of native carotenoids (according to the USDA database and own blank examinations), was purchased from a local supermarket (Delhaize, Strassen) and used without further purification. 2.2 β-Carotene, WPI and enzyme solutions β-Carotene standard solution was first prepared by dissolving 1 mg of β-carotene standard in 2 mL of hexane. The solution was sonicated for 5 min. and warmed up at 30 °C for 5 min. (Ultrasonic Cleaner, VWR Symphony ® , Massachusetts, USA). Then, 1 mL of peanut oil was added to foster the Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 7 dissolution of β-carotene by sonication and warming at 30 °C for 5 min. The hexane was removed by evaporation under a stream of nitrogen for ± 20 min. at 30 °C (TurboVap LV from Biotage ® , Uppsala, Sweden). The standard β-carotene solution was made by the addition of 4 mL of peanut oil to the remaining 1 mL to reach a concentration of 0.2 mg/mL, and two other concentrations (0.1 and 0.4 mg/mL) were also prepared for further investigation by adding 9 and 1.5 mL (instead of 4 mL) of peanut oil, respectively. The concentration of the standard solutions was later verified spectrophotometrically as explained below (section 2.5). Aliquots of the standard solutions were pipetted into 15 mL falcon tubes, and stored at -80 °C until usage.

Assuming a total volume of 2 L of intestinal fluids during GI digestion, the amount of β-carotene added to each digesta was 30 μg, by pipetting 75, 150 or 300 μL from the standard solutions (0.4, 0.2 or 0.1 mg/mL, respectively), representing 1.2 mg/L of β-carotene and 3, 6 or 12 g/L of oil (Table 1).

In the same way, the amounts of WPI tested were 0, 7.5 and 15 g/L (Table 1), corresponding to 0, 25 and 50% of the recommended dietary allowance (RDA) (60 g/d for human adults) [START_REF] Hautvast | Recommended dietary allowances for Europe[END_REF]. Practically, 0, 187.5 or 375 mg of WPI were dissolved in 6.25 mL of pure water, and together with the added β-carotene solution in oil, the final ratio of matrix (WPI+β-carotene solution) to simulated gastric fluid of 50:50 (v/v) was reached. A brief mixing of the WPI solution with the beta-carotene in peanut oil and the simulated gastric fluid (SGF) was achieved by vortexing for 15 sec. SGF and the simulated intestinal fluid (SIF) were made as recommended by the European consensus digestion model [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]. Pepsin solution was prepared in SGF to reach a final concentration of 2000 U/mL of the final gastric mixture. In addition to the standardized concentration (Table 1), two other concentrations were tested (1000 and 4000 U/mL).

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 8 Regarding pancreatin and bile extract, pancreatin was weighed based on a trypsin activity of 100, 200 or 400 U/mL, while the bile extract was weighed in order to reach a concentration of 3.4, 6.8 or 13.6 mg/mL of the final digestion volume (Table 1); both were prepared in the same SIF solution.

It is noteworthy that the European consensus suggested a standardized amount of pancreatin based on trypsin activity at 100 U/mL, but in the present study, pancreatin amount was doubled following pre-experiments (200 U/mL of trypsin), in order to enhance the digestibility of both WPI and oil (by increasing at the same time the lipase activity). As explained above, two other concentrations of pancreatin (100 and 400 U/mL) as well as bile extract (3.4 and 13.6 mg/mL) were tested. Conditions with low pancreatin (100 U/mL) and/or low bile (3.4 mg/mL) are denoted in the following as marginal digestive conditions in this investigation.

Simulation of gastro-intestinal (GI) digestion

In vitro simulated GI digestion was carried out according to the harmonized INFOGEST protocol, with some modifications as explained above (section 2.2). The model was used to test the influence of proteins at three different concentrations on β-carotene bioaccessibility, including also rather marginal, i.e. insufficient digestion parameters. The oral phase was omitted as the matrix employed did not include significant amounts of carbohydrates and was liquid.

For the gastric phase, 6.25 mL of protein solution at the desired concentration (equivalent to 0, 25 or 50% of the RDA) was added to each sample, followed by the addition of 75, 150 or 300 μL of βcarotene solution in oil (corresponding to a total of 30 μg of β-carotene per digest, with amounts similar to former in vitro experiments [START_REF] Biehler | Divalent minerals decrease micellarization and uptake of carotenoids and digestion products into Caco-2 cells[END_REF]. Before the addition of 4 mL of SGF (1.25 x concentrate), 1 mL of pepsin solution was added to the mixture in order to reach enzymatic activities of 1000, 2000, 4000 U/mL of the final gastric volume. Then, 31 μL of calcium dichloride (0.03 M) was added to reach a concentration of 0.075 mM in the final mixture, and finally, pH

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 9 adjustment to 3 by hydrochloric acid (1 M) was carried out, bringing the volume of each sample to 12.5 mL with pure water.

Finally, the samples were incubated in a shaking water bath (GFL 1083 from VEL ® , Leuven, Belgium) for 1 or 2 h at 37 °C, with a shaking speed of 75 or 100 rounds per minute (equal to a doubling of the kinetic energy, which is proportional to the square of the speed), depending on the desired condition. In the standardized INFOGEST digestion model [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF], no specific shaking speed was suggested.

At the end of the gastric incubation, 7.5 mL of SIF, and 2.5 mL of pancreatin and bile extract solution at various concentrations (100, 200 or 400 U/mL based on trypsin activity for pancreatin, and 3.4, 6.8 or 13.6 mg/mL for the bile extract) were added to the chyme. Then, 250 μL of calcium dichloride (0.03 M) was added to reach a concentration of 0.3 mM in the final mixture. Before bringing the volume to 25 mL, the pH was adjusted to 7 by the addition of sodium hydroxide solution (1 M), then the samples were incubated for 2 h at 37 °C, with a shaking speed of 75 or 100 rounds per minute (rpm).

β-Carotene extraction

At the end of the intestinal incubation, 12 mL were removed from the digesta and transferred into 15 mL falcon tubes. The samples were centrifuged for 1h at 3200 x g (4 °C), then 5 mL were collected from the middle aqueous phase and filtered through 0.2 μm nylon membrane syringe filter. In a new 15 mL falcon tube, 4 mL of hexane:acetone (2:1, v:v) were added to 2 mL of the filtered aqueous phase, vortexed for 30 sec. and centrifuged for 2 min. at 3200 x g (4 °C). The supernatant, which represents the hexane phase, was transferred into a new falcon tube, then the extraction process was repeated two times with 4 mL of hexane. All extracts were combined in the same tube and dried under a stream of nitrogen and stored at -80 o C until analysis.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 10 2.5 Spectrophotometric analysis Spectrophotometric analysis of β-carotene was done as described previously [START_REF] Corte-Real | Selective factors governing in vitro betacarotene bioaccessibility: negative influence of low filtration cutoffs and alterations by emulsifiers and food matrices[END_REF]. The dried residue was reconstituted in 1 mL of cold (4 o C) hexane and transferred to a 1 mL quartz cuvette (absorption cuvette, Hellma®, New York, USA).

The absorbance was measured between 300 and 600 nm (GENESYS TM 10S UV-Vis Spectrophotometer, Thermo Fisher Scientific, Massachusetts, USA). The concentration was calculated by applying the Beer-Lambert law, taking into account the molar absorption coefficient [START_REF] Britton | Carotenoids Handbook[END_REF], and different dilution steps:

where C = concentration of the measured solution (mg/mL), abs max = maximum measured absorbance of β-carotene at approx. 450 nm, Fd = dilution factor adjusting for extraction or reconstitution process, ε λ = molar absorption coefficient (Lmol -1 cm -1 ) of β-carotene in hexane (138824), d = length of light pathway through cuvette (cm) and M = β-carotene molar mass (536.89 g/mol).

Finally, the percentage of β-carotene micellization was used as a measure of bioaccessibility, and was expressed as the percentage of the solubilized amount of β-carotene present in the aqueous phase of the filtered digesta after in vitro GI digestion, compared to the initial amount added to the sample.

Further physico-chemical characterization of digesta

To study further physico-chemical properties of the digesta, macroviscosity and surface tension, micelle size and zeta potential, fatty acid titration, and the emulsion structures were investigated, as these parameters were also believed to possibly impinge on β-carotene bioaccessibility.

Rheological measurements and surface tension of the digesta

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 11 Flow curves were determined as a function of the shear rate between 0.1 s -1 and 130 s -1 on an Anton-Paar rheometer (MCR 302, WESP, Graz, Austria), equipped with a double gap cell at 5°C. Each measurement cycle did comprise a temperature equilibration phase, a pre-shear phase at 5 s -1 of 60 s, a flow curve with increasing shear rates, an intermediate equilibration phase at 131 s -1 of 60 s and a flow curve with decreasing shear rates. At the end of each cycle, a regression flow curves was calculated for the shear rate range between 0 s -1 and 100 s -1 for the shear stress respectively between 1 s -1 and 100 s -1 for the viscosity. Each measurement cycle was performed three times, because of the fluidity of the samples. The regression curves were averaged and plotted as viscosity and shear stress as a function of the shear rate.

Surface tension of digesta samples, pre-conditioned at 25 ± 0.1 °C, were determined by the weight-drop method as previously described by [START_REF] Permprasert | Evaluation of the effects of some additives and pH on surface tension of aqueous solutions using a drop-weight method[END_REF]. The air-water interfacial properties of digesta were calculated as follows:

H2O

where σ H2O = 71.99 dyn cm -1 is the surface tension of pure water.

Micelle size and zeta potential analysis

Aliquots of the aqueous micellar fraction obtained after GI digestion under standard conditions (Table 1) were filtered through a 0.2 μm nylon membrane syringe filter for the analysis of the micelle size and zeta potential. The intensity-weighted mean hydrodynamic radius and zeta potential were determined by dynamic light scattering and laser doppler micro-electrophoresis, respectively. Measurements were done at room temperature with at least four replicates, by using a Zetasizer Nano Zs instrument (Malvern Instruments, Malvern, UK).

Lipid and protein digestion

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003

12

The amount of free fatty acids in the digesta after GI digestion was estimated as described previously [START_REF] Corte-Real | Magnesium affects spinach carotenoid bioaccessibility in vitro depending on intestinal bile and pancreatic enzyme concentrations[END_REF], based on the titration against sodium hydroxide (NaOH 0.1 M), using phenolphthalein as an indicator.

Sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) was carried out to study completeness of protein digestion. Samples were denatured at 95 o C for 5 min. in Lämmli buffer.

About 22 µg of protein per pocket was loaded to a 15% acrylamide and 0.4% bisacrylamide gel (gastro-intestinal digestion) and 45 g of protein per pocket was loaded to a 10% acrylamide/0.3% bisacrylamide gel (gastric digestion). Marker was Invitrogen novex see blue plus 2 (Invitrogen, Carlsbad, CA). Gel was run at 80 V (30 min.) plus 100 V (90 min.). Running buffer contained SDS (0.1%), glycine (190 mM), tris-HCl (25 mM). Fixation/staining was done with methanol/acetic acid/coomassie brilliant blue R (50%, 10%, 0.1%) (30 minutes), destaining in methanol/glacial acetic acid/water (30%, 10%, 60%, 3 h). Pictures were taken in a Kodak Gel Logic 2200 Imaging System (Kodak, Rochester, NY).

Confocal laser scanning microscopy (CLSM)

Confocal imaging of emulsion structures before and after GI digestion under standard conditions (Table 1) was carried out at room temperature with a confocal laser scanning microscope (Zeiss LSM 880, Airyscam SR, Jena, Germany), using a 63x objective. Nile Red dissolved in ethanol and FITC dissolved in acetone were mixed into the aliquots of digesta for fat and protein staining with final concentrations in the samples of 1 µg/mL. A small-sample aliquot was placed on a slide for visualization. The fluorescent dyes were excited by an Argon 488 nm laser and the emitted light was collected at 522 nm for proteins and 635 nm for the fat phase. The diameter of lipid droplets (n=20/group) at various concentration of WPI was measured by using a modular image-processing and analysis software for digital microscop (Zen 2.3 blue edition, Carl Zeiss Microscopy GmbH, Jena, Germany).

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 13 2.7 Statistical analyses and data treatment In order to minimize day-to-day variations between experiments, bioaccessibility of β-carotene was normalized to a daily control which was assessed for each digestion. Unless otherwise stated, all values are expressed as the mean ± standard deviation. Replicates were obtained from at least 2 individual sets of analyses obtained at different days (N≥2), and at least 4 replicates were obtained for each digestive condition during 1 set of analyses.

Statistical analysis was performed using SPSS 22 software (SPSS Inc., Chicago, IL). Normal distribution of data was verified by Q-Q -plots and equality of variance by box-plots and Levene's test. For statistical evaluation, β-carotene bioaccessibility values were log-transformed to achieve a normal distribution. Linear mixed models were developed with the effect of WPI concentration, amount of lipid source, bile extract concentration, kinetic energy and duration of gastric phase, pepsin and pancreatin concentration as fixed factors and log-β-carotene bioaccessibility as the observed dependent factor. As a model including all factors was not fully factorial, but several significant interactions were obtained, additional linear mixed models were run to keep constant certain parameters and to better allow further group-wise comparisons. P-values < 0.05 were considered statistically significant different (2-sided). Where needed, Fisher F-tests were followed by post hoc tests (Bonferroni, for comparing >3 groups) or LSD tests (for comparison of ≤ 3 groups).

Results

Interactions of WPI with matrix related effects affecting β-carotene bioaccessibility

3.1.1 Bioaccessibility under standard conditions and with escalating concentrations of WPI Under control conditions (no addition of WPI), bioaccessibility of pure β-carotene following the GI digestion averaged approx. 0.3 μg/mL, representing around 25% of β-carotene recovered from Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 14 the aqueous micellar fraction at the end of the in vitro digestion, compared to the initial amount added at the beginning of digestion (Fig. 1A). Although the addition of an equivalent of 50% protein RDA of WPI slightly enhanced bioaccessibility (Fig. 1A) compared to the control (no protein added), this effect was not significant under standard conditions [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF].

Influence of WPI at different amounts of co-digested oil on β-carotene bioaccessibility

Following linear mixed models, the effect of oil (p<0.001) was statistically significant, while the influence of WPI had no significant effect. In addition, a significant interaction between the amount of added oil and WPI concentration was encountered (p<0.001), thus the effects were further studied at individual WPI levels.

When no WPI was added, increasing the volume of oil (up to 300 μL) negatively affected the bioaccessibility (p<0.001) compared to the standard conditions (150 μL of oil). No significant effect on β-carotene bioaccessibility was observed with a lower amount of oil (75 μL of oil, Fig. 1B, Table 2).The addition of 25% RDA of WPI to samples with a reduced amount of oil further enhanced the bioaccessibility (by up to 20%) compared to the control, while the addition of WPI at 50% RDA had no significant effect (Fig. 1B). However, adding WPI to the simulated GI digestion with a high volume of oil (300 L) caused an additional decrease in bioaccessibility, in a dose dependent manner (p<0.001). In this scenario, adding the equivalent of 25 and 50% RDA of WPI significantly decreased β-carotene bioaccessibility by 20% (p=0.017) and up to 35% (p<0.001), respectively, compared to the control (no WPI).

3.2 Influence of WPI on β-carotene bioaccessibility at various gastric conditions 3.2.1 Impact of WPI at altered simulated peristalsis on β-carotene bioaccessibility Following linear mixed models, the effect of peristalsis conditions on β-carotene bioaccessibility was statistically significant (p<0.001), while the influence of WPI had no significant effect. Also, Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 15 the interaction between the added amount of WPI and peristalsis movements had a significant (p=0.019) effect on the bioaccessibility of β-carotene, thus effects were further studied at individual shaking speed.

The higher shaking water speed of 100 rpm induced an up to two times higher β-carotene micellization (p<0.001) compared to the lower speed of 75 rpm (Fig. 2, Table 2), across all 3 levels of proteins present (0, 25 or 50% of RDA), at 2 h of gastric digestion time.

Following all-group-wise comparisons (post hoc-tests), the addition of 25% RDA of WPI significantly increased (p<0.001) β-carotene bioaccessibility when a higher kinetic energy was applied, relative to the control (Fig. 2A). However, no significant influence was observed with the addition of 50% RDA of WPI, i.e. WPI did not influence β-carotene bioaccessibility in a linear dose-dependent manner.

Influence of WPI on β-carotene bioaccessibility at altered gastric phase duration

Following linear mixed models, the effect of gastric phase duration (p<0.001) was statistically significant, while the influence of WPI had no overall significant effect. However, the impact of gastric digestion time (2 vs. 1 h) depended significantly on the shaking speed (p=0.0016, Fig. 2A), with larger differences observed at lower simulated peristalsis.

Reducing gastric transition to 1 h decreased bioaccessibility by 25% at 75 rpm (in general for all levels of WPI addition), while the negative effect was less pronounced, yet significant, with a higher shaking speed, compared to the standardized 2 h of gastric phase (p<0.001, Table 2).

At 75 rpm, no further influence was noticed on β-carotene bioaccessibility following the addition of WPI, with a reduced gastric digestion time (Fig. 2A), while a significant (p<0.001) interaction was found at 100 rpm between protein concentration and gastric phase duration (Fig. 2A). Here, the addition of WPI significantly (p<0.001), though only slightly, increased the bioaccessibility of βcarotene by up to 20% (for the 25% RDA protein group), compared to the control.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 16

Effect of varying concentrations of pepsin

Following linear mixed models, the effects of both WPI (p<0.001) and of pepsin concentration (p<0.001) were statistically significant. A statistical significant interaction of pepsin x protein (p=0.008) was also found regarding β-carotene bioaccessibility.

Under control conditions (no WPI), no influence of varying the amount of pepsin on β-carotene micellization (Fig. 3A) was found. Similarly to the standard conditions (2000 U/mL of pepsin), addition of WPI to the simulated GI digestion with 4000 U/mL of pepsin did not influence βcarotene bioaccessibility compared to the daily control (Table 2). However, the addition of WPI to the samples with a lower concentration of pepsin decreased β-carotene bioaccessibility by up to 40% (p<0.001), compared to the control (no WPI).

Influence of WPI at altered intestinal conditions

Effect of varying concentrations of bile extract

Following linear mixed models, the effects of both WPI (p=0.002) and of bile concentration (p<0.001) were statistically significant. In addition, a significant interaction between added amounts of bile extract and protein concentration was found (p<0.001).

A maximum of β-carotene micellization (around 25%) was obtained with the higher amounts of bile extract (6.8 and 13.6 mg/mL), while 3.4 mg/mL of bile extract significantly (p<0.001) decreased β-carotene bioaccessibility, compared to the standard conditions (6.8 mg/mL, Fig. 3B).

There was no significant effect of adding WPI at high amounts of bile (6.8 and 13.6 mg/mL per digesta), except for a slight decrease of β-carotene bioaccessibility when 50% RDA of WPI was added to the samples with highest amounts of bile extract (p=0.0011). However, the addition of WPI to the digesta with reduced amount of bile extract (3.4 mg/mL) negatively affected the bioaccessibility in a concentration dependent manner (p<0.001). The addition of 25% and 50%

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 17 RDA of WPI decreased the bioaccessibility to half and 1/3, respectively, compared to the control (p<0.001, Table 2).

Effect varying concentrations of pancreatin

Following linear mixed models, the effects of both WPI amount (p=0.009) and of pancreatic enzyme concentrations (p<0.001) were statistically significant. Under control conditions (no WPI), increasing the pancreatin concentrations positively affected the bioaccessibility (p<0.001) in a concentration dependent manner (Fig. 2B), i.e. increasing β-carotene micellization from 25% up to 34% (75 rpm), and from 45% up to 68% (100 rpm).

Furthermore, a statistical interaction (pancreatin x peristalsis x protein, p=0.004) regarding βcarotene bioaccessibility was also found. At 75 rpm, the addition of WPI to the simulated GI digestion with half the typical amount of pancreatin negatively affected (p<0.001) β-carotene bioaccessibility, by up to 50%, compared to the control (Fig. 2B, Table 2), in a dose-dependent fashion. When doubling the amount of pancreatin to 400 U/mL, a significant (p<0.001) negative effect of WPI on β-carotene micellization was found only with the addition of 25% RDA, compared to the control.

A similar effect was observed for conditions resembling a stronger peristalsis effects (100 rpm).

Using the double amount of pancreatin (400 U/mL) slightly decreased β-carotene micellization following the addition of 50% RDA of WPI (p=0.006, Fig. 4). However, employing half the amount of pancreatin compared to standard digestive conditions resulted in ambivalent effects, with 25% RDA of WPI resulting in enhanced β-carotene bioaccessibility (p=0.004), while the addition of 50% RDA of WPI slightly decreased β-carotene bioaccessibility (p=0.015), compared to the control.

Effect of WPI on the physico-chemical properties of the digesta

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003

18

In general, the shear stress and viscosity of digested samples at various concentrations of WPI was found very close to that of aqueous systems (Supplementary Fig. 1A, left panel), and samples had a very small yield point (0.01 Pa or even lower), with lower yield points after digestion. Nondigested WPI solutions at 0% RDA and all digested solutions showed almost Newtonian behaviour above shear rates of 5 s -1 (Supplementary Fig. 1A, right panel).

Surface tension for control and supplemented digesta with 25% of WPI showed no significant difference (Supplementary Fig. 2). The addition of 50% WPI slightly reduced surface tension compared to the control digesta (p<0.001), being in line with the higher macroviscosity data.

Regarding mixed micelle size and zeta potential, both 25% and 50% RDA of WPI additions resulted in significantly reduced micelle size of 3.3 ± 0.1 nm compared to WPI free control digesta (supplementary Fig. 3A, p<0.001). However, the average absolute zeta potential of the samples digested with 25% and 50% RDA of WPI was somewhat reduced (from -46.8 ± 1 mV to -25.3 ± 6.2 and -28.8 ± 1.7 mV, respectively (Supplementary Fig. 3B, p<0.001).

Lipid digestion results obtained by fatty acid titration were not fully informative and rather reflected the buffering capacity of the added proteins, and/or additional protein hydrolysis, thus it was not feasible to separate their contribution to lipolysis (Supplementary Fig. 4). The size of lipid droplets and the co-location of lipids and proteins at 25% and 50% RDA samples were measured by confocal microscope. Prior to digestion, the lipid droplets were distributed as large aggregates of lipid droplets in the mixture. With increasing concentration of WPI, the lipid droplets formed smaller aggregates in the digesta (Fig. 4A). The higher concentration of WPI resulted in a smaller average lipid droplet size compared to the control digesta, decreasing from 13.42 ± 9.28 to 4.84 ± 1.52 and to 3.35 ± 1.76 µM for 0, 25 and 50% WPI, respectively (Supplementary table I), with the first group being significantly different from the last 2 groups (p<0.001).

Regarding protein digestion, there was no detectable difference between the various digestive conditions on the WPI originating bands detectable by SDS-PAGE, following either gastric or Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 19 gastro-intestinal digestion (Supplementary fig. 5, upper panel). Gastro-intestinal digestion suggested the complete digestion of WPI into smaller fragments during all digestive conditions, as no major bands were visible following digestion. Only bands originating from enzyme additions were detectable after gastro-intestinal digestion. Following only gastric digestion however (Supplementary fig. 5, lower panel), fragments around 18 kDa were clearly visible after WPI digestion, for all digestive conditions, likely representing either α-lactalbumin (14.2 kDa) and or βlactoglobulin (18.4 kDa).

Discussion

Micellization of β-carotene constitutes one of the major steps determining its absorption and bioavailability, and previous studies have shown that they are well correlated [START_REF] Biehler | Methods for assessing aspects of carotenoid bioavailability[END_REF][START_REF] Reboul | Bioaccessibility of carotenoids and vitamin E from their main dietary sources[END_REF]. Several reports have highlighted matrix-related as well as host-related factors influencing carotenoid bioaccessibility and absorptive process, as reviewed earlier [START_REF] Bohn | Bioavailabilty of non-provitamin A carotenoids[END_REF][START_REF] Bohn | Host-related factors explaining interindividual variability of carotenoid bioavailability and tissue concentrations in humans[END_REF]. In this regard, proteins have been proclaimed to aid in emulsifying liposoluble dietary constituents [START_REF] Soukoulis | A comprehensive overview on the micro-and nano-technological encapsulation advances for enhancing the chemical stability and bioavailability of carotenoids[END_REF], but their presence has also shown a negative influence on lipid degradation, which could have an impact on the micellization process [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF]. In the present study, we investigated the effect of WPI, a commonly consumed protein, on β-carotene bioaccessibility under selective simulated digestion conditions to study its influence at more physiological vs. somewhat sub-optimal conditions regarding lipid and protein digestion.

The most important finding was that the influence of WPI on β-carotene depended on digestive conditions. More specifically, WPI reduced β-carotene bioaccessibility by approx. 70% when lipid digestion was rather compromised by lower concentrations of bile, enzymes, or when reducing shaking speed. However, under certain conditions, enhanced bioaccessibility, compared to standard digestion conditions (European consensus model, [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]), of up to 20%, were also Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 20 encountered, but appeared less consistent. Under standard digestion conditions (Table 1, Fig. 1A), adding WPI reflecting 0, 25 and 50 % of the RDA for a 70 kg adult (assuming a digestion volume of 2 L) during simulated GI digestion, had no significant effect on the micellization of β-carotene, in spite of resulting in lower lipid droplet size after GI digestion. To our knowledge, this is the first report systematically investigating carotenoid bioaccessibility in the presence of a co-digested protein, under varying simulated digestion conditions.

Whey protein consists primarily of β-lactoglobulin (50-55%) and α-lactalbumin (20-25%).

Regarding its emulsion stabilizing capability, it was emphasized that the high percentage of βpleated sheet (~50%) conformation and the relatively low content of the α-helix form (~15%) allows β-lactoglobulin (due to its higher hydrophobicity) to adsorb and produce better conformational changes at the o/w interface, compared to α-lactalbumin (β-sheet ~ 9%, α-helix ~ 45%) [START_REF] Carbonaro | Relationship between digestibility and secondary structure of raw and thermally treated legume proteins: a Fourier transform infrared (FT-IR) spectroscopic study[END_REF][START_REF] Schröder | Interfacial properties of whey protein and whey protein hydrolysates and their influence on O/W emulsion stability[END_REF]. In addition, during the gastric phase, a complete hydrolysis of α-lactalbumin was found, while a portion of β-lactoglobulin remained resistant after 1 h [START_REF] Malaki Nik | Impact of interfacial composition on emulsion digestion and rate of lipid hydrolysis using different in vitro digestion models[END_REF]. Thus, in whey protein-stabilized emulsions, β-lactoglobulin may play a greater role regarding emulsion stability, compared to α-lactalbumin.

The strongest positive significant effects of WPI addition were observed when halving the amount of oil (Fig. 1B) and doubling the peristalsis energy (Fig. 2A), reflecting more efficient digestive conditions. The effects were an approx. 20% increase, compared to their respective controls (no WPI addition), but only at protein concentrations equivalent to 25% RDA, not 50% RDA. It was reported earlier that β-lactoglobulin can interact with β-carotene within a pocket of hydrophobic residues, with strong non-covalent interactions [START_REF] Mensi | Interactions of betalactoglobulin variants A and B with Vitamin A. Competitive binding of retinoids and carotenoids[END_REF], suggesting that WPI can act as a vehicle for hydrophobic compounds during the GI digestion. This may explain the positive effect observed when reducing the amount of oil. Such carotenoid-protein interactions are not an isolated case. For instance, orange carotenoid-Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 21 protein complexes with glutathione-S-transferase and crustacyanin are examples of well characterized carotenoid-binding proteins [START_REF] Pilbrow | Carotenoid-binding proteins; accessories to carotenoid function[END_REF]. In addition, native whey proteins are highly surface-active molecules [START_REF] Mun | Fluorescence imaging of spatial location of lipids and proteins during digestion of protein-stabilized oil-in-water emulsions: A simulated gastrointestinal tract study[END_REF]. It was demonstrated that the proteins and peptides liberated during GI digestion can affect the stability of o/w emulsions (van der Ven, Gruppen, [START_REF] Van Der Ven | Emulsion Properties of Casein and Whey Protein Hydrolysates and the Relation with Other Hydrolysate Characteristics[END_REF], as well as retarding triglyceride oxidation [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF]. It was also reported that after gastric digestion, some of the protein fragments remained adsorbed to the lipid droplet surfaces.

However, these appeared to be removed into the surrounding aqueous phase following further proteolysis by pepsin, the low pH, and peristalsis movements in the stomach, as well as further action of the bile salts in the small intestine [START_REF] Mun | Fluorescence imaging of spatial location of lipids and proteins during digestion of protein-stabilized oil-in-water emulsions: A simulated gastrointestinal tract study[END_REF], allowing access to lipolytic enzymes. This is in line with results from the present study, as no more adsorption of proteins to lipid droplets in fully digested samples was seen by confocal microscopy.

These considerations are in line with the present outcomes (Fig. 2A) and the positive influence of peristalsis movements on the bioaccessibility.

Contrarily, the higher dose of WPI (reflecting 50% RDA) resulted in reducing bioaccessibility to the level of the standard conditions (Fig. 2B). We can only speculate for the reasons. It is possible that a higher amount of protein, perhaps in conjunction with a less complete digestion, resulted in liberating large peptides at the o/w interface, which may obstruct the displacement of WPI from lipid droplet surfaces and/or hindering digestive enzymes to adsorb to the droplet surfaces as reported previously [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF]. In general, the negative effects of WPI were more pronounced (reducing bioaccessibility by up to 70%) and consistent compared to the positive effects, and were observed especially at conditions reflecting incomplete lipid digestion (low amount of bile and/or pancreatin, or high content of oil).

It is proposed, as supported by the observed smaller lipid droplet size with higher protein addition (Fig. 4), that emulsion stability is influenced, in addition to the presence of emulsifiers, by the oil Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 22 droplet size, and by the formation of a viscoelastic film at the oil droplet interface, formed by peptides liberated during the GI digestion of whey proteins [START_REF] Schröder | Interfacial properties of whey protein and whey protein hydrolysates and their influence on O/W emulsion stability[END_REF]. In general, it is accepted that smaller lipid droplet size enhances lipid digestion and thus carotenoid micellization [START_REF] Salvia-Trujillo | Influence of particle size on lipid digestion and beta-carotene bioaccessibility in emulsions and nanoemulsions[END_REF], though in this study, we were unable to measure the effect of proteins on lipid digestion due to the over-layering effect of protein buffering capacity and/or hydrolysis. Also, it was also shown earlier that the generated peptides strongly affected the stability of the o/w emulsions during the gastric phase, dictating the resulting size of lipid droplets [START_REF] Malaki Nik | Impact of interfacial composition on emulsion digestion and rate of lipid hydrolysis using different in vitro digestion models[END_REF]. Thereupon, WPI-coated droplets, at least during earlier stages of digestion, may hinder the access of pancreatic lipase (and pancreatic co-lipase) to the droplet surface, especially at low bile salt concentration, due to the large and/or compact peptide film adsorbed to the lipid droplet surfaces [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF]. In our study, this effect was corroborated by lower amounts of pepsin (Fig. 3A), and perhaps a less complete protein digestion, though this could not be differentiated in the present SDS-page (supplementary fig. 5).

However, protein digestion following gastric phase was found to be incomplete, with remaining bands of α-lactalbumin and/or β-lactoglobulin, which may have restricted the subsequent adsorption of lipase required for lipid digestion. This could have resulted in the observed decrease in βcarotene bioaccessibility (by 40%), which was not observed with regular or elevated amounts of pepsin (2000 or 4000 U/mL, respectively).

Likewise in line, reducing the amount of pancreatin (Fig. 2B) or doubling the amount of oil (Fig. 1B) resulted in negative effects on β-carotene bioaccessibility, by up to 50%. Pancreatin contains lipase, allowing for fat hydrolysis, and either doubling the amount of oil, or halving the amount of pancreatin could cause a relative lack of lipolytic enzymes, thereby promoting incomplete droplet processing, flocculation and coalescence [START_REF] Qiu | Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein[END_REF]. The addition Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 23 of WPI further impeded the bioaccessibility in a dose-dependent manner, decreasing β-carotene bioaccessibility by half with a higher dose of WPI, compared to the control conditions.

Finally, halving the amount of bile (Fig. 3B) resulted in one of the most drastic negative effects on β-carotene bioaccessibility, with the addition of WPI reducing bioaccessibility of β-carotene to one third (Fig. 3B). The results obtained by Nik et al. showed that WPI undergoes further displacement from lipid droplet surfaces in the presence of other surface-active compounds, such as bile salts during the intestinal phase, offering finally access to lipolytic enzymes [START_REF] Malaki Nik | Impact of interfacial composition on emulsion digestion and rate of lipid hydrolysis using different in vitro digestion models[END_REF]. In their study, this induced a higher degree of lipolysis and led to the formation of a smaller lipid droplets. This suggests that, in addition to proteolytic enzymes and peristalsis movements, the presence of bile at the interfacial composition of the original emulsion plays a major role in determining the extent of lipolysis.

The additional physico-chemical properties investigated following GI digestion under standard conditions yielded additional insights regarding potential effects of WPI on digestive processes and carotenoid bioaccessibility. Firstly, the addition of proteins did not have a marked effect on the macro-viscosity or surface tension of the digesta, precluding that changes in these parameters effected bioaccessibility. This is further corroborated by the small changes in sheer-forces, and in line with a rather complete hydrolysis of added proteins following GI digestion to smaller, non SDS-PAGE detectable fragments, under all digestive conditions (suppl. Fig. 5). In the future, it would be interesting to follow the fate of smaller protein fragments, e.g. by LC-MS-MS, which was not carried out in the present study. However and secondly, protein addition, via their emulsifying properties, reduced lipid droplet size after digestion, and also resulted in lower mixed micelle size, which may have contributed to the positive effect on carotenoid bioaccessibility. It is unclear why the absolute zeta-potential was somewhat decreased with higher protein addition, suggesting a less stable emulsion, though effects could have contributed to the mixed effects encountered.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003

24

In summary, the addition of WPI during simulated GI digestion influenced the bioaccessibility of β-carotene positively as well as negatively, depending on the digestion conditions. The results obtained in this study are in line with previous findings regarding the ability of WPI to adsorb to lipid droplet surfaces and playing a major role in stabilizing o/w emulsions, dictating the fate of lipophilic compounds. In this study, the ability of WPI to improve β-carotene bioaccessibility was somewhat limited (around 20%), and found when applying a higher peristalsis energy (rather more suggestive of a healthy situation), or when reducing dietary lipids. The negative effects resulted following halving the amount of enzymes (pancreatin and pepsin) or doubling the amount of oil, and halving the amount of bile, decreasing the bioaccessibility of β-carotene up to one third after the addition of WPI. These results suggest that WPI and possibly other proteins may modulate carotenoid bioaccessibility, likely via their interaction during the processing of lipid droplets into mixed micelles during digestion as suggested by the altered droplet sizes found. When hypothetically transferring results to humans, a negative influence of WPI on β-carotene may be expected when digestion is less complete as characterized by lower enzyme secretion, while under rather normal and healthy conditions no negative effects may occur. Finally, a potential influence of WPI on other carotenoids (such as xanthophylls), and/or the influence of other proteins on βcarotene remains to be shown, as well as the possible influence of the food-matrix on proteincarotenoid interactions, and importantly, a confirmation of such in vitro results by in vivo studies.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003

25

The help of Bernadette Leners during digestions, fatty acid titrations and SDS-page is much appreciated.

The authors declare that there are no conflicts of interest. 51.9 ± 0.7 B 58.7 ± 1.3 B 65.9 ± 3.9 A 12.7 ± 1.9 C 30.6 ± 2.8 A 33.3 ± 2.2 A 40.8 ± 1.4 C 49.3 ± 2.5 A 58.2 ± 3.9 B Bile extract 3.4 mg/mL I 6.8 mg/mL II 13.6 mg/mL II 21.1 ± 0.9 A 24.8 ± 2.5 A 25.5 ± 0.7 A 10.0 ± 0.7 B 24.1 ± 3.3 A 26.5 ± 1.1 A 7.5 ± 0.9 C 30.6 ± 2.8 A 22.6 ± 0.7 B RDA: recommended dietary allowance. 1 Bioaccessibility is expressed as the percentage of βcarotene recovered at the end of the in vitro GI digestion, compared to the amount added at the beginning of digestion. 2 Concentrations given in final digestion stage (small intestine) are 0, 25 and 50% based-RDA.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y., [START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 31 ----------------------------+--------------------------100 rpm -------------------

  Unless otherwise specified, all chemicals were of analytical grade or superior. Potassium chloride (≥99%, Art. No. P9541), potassium phosphate (monobasic ≥99, Art. No. P5655), sodium bicarbonate (≥99, Art. No. S5761), sodium chloride (≥99.5%, Art. No. 71376), magnesium chloride hexahydrate (Art. No. M2393), ammonium carbonate (Art. No. 68392), sodium hydroxide solution (1 M, Art. No.1.09137), calcium chloride dihydrate (≥99, Art. No. C3306), nile red (Art. No. 19123), fluorescein isothiocyanate isomer I (FITC) (Art. No. F7250), and phenolphthalein (Art. No. 105945) were acquired from Sigma-Aldrich. Hexane (≥95%, Art. No. 24574), acetone (≥99%, Art. No. 20063), and hydrochloric acid (1 M, Art. No. 30024.290) were obtained from VWR (Leuven, Belgium).

  M.I. conducted the majority of the analyses and wrote the first version of the manuscript. T.B. designed the trials and aided in statistical evaluation and manuscript preparation. C.D. conducted a large number of digestion experiments. Y.L. participated in the design of the study and the final manuscript version. T.S. conducted viscosity measurements, G.D. performed surface tension and lipid titration experiments. C.D. and P.B. conducted the analyses of zeta potential and micelle size.

Fig. 1 .

 1 Fig. 1. Influence of whey protein isolate (WPI) (A), and oil, co-digested with WPI (B) on β-

Fig. 2 .Fig. 3 .

 23 Fig. 2. A) Influence of peristalsis energy and gastric phase duration on β-carotene

Fig. 4 .Highlights

 4 Fig. 4. Distribution of lipid droplets in the presence and absence of whey protein isolate (WPI),

  Figure 3

Table 1 .

 1 Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y.,[START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y.,[START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y.,[START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 Overview of digestion conditions studied in dependence of various concentrations of whey protein isolate (WPI).

	29

$ all concentrations are per final volume of digesta at the respective digestive step *Termed standard conditions , according to the European consensus digestion model

[START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]

, except RPM which was not defined.

Comment citer ce document : Iddir, M., Degerli, C., Dingeo, Desmarchelier, C., Schleeh, T., Borel, P., Larondelle, Y.,

[START_REF] Degerli | Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions[END_REF]

. Whey protein isolate modulates beta-carotene bioaccessibility depending on gastro-intestinal digestion conditions. Food Chemistry, 291, 157-166 . , DOI : 10.1016/j.foodchem.2019.04.003 30

Table 2 .

 2 Average bioaccessibility (%) 1 of pure β-carotene digested with whey protein isolate (WPI) at varying concentrations 2 . Values represent mean ± SD of n = 4 and N≥2 (sets repeated at different days). Means without a common superscript differ significantly, P<0.05.

			Bioaccessibility (%)
			WPI (% RDA)
	Condition	0	25	50
	Oil 3 g/L I 6 g/L I 12 g/L II	21.4 ± 2.2 A 24.8 ± 2.5 A 20.7 ± 1.2 A	25.6 ± 0.8 B 24.1 ± 3.3 A 16.8 ± 1.2 B	22.7 ± 0.9 A 30.6 ± 2.8 A 13.5 ± 0.8 C
	Peristalsis energy 75 rpm I 100 rpm II	24.8 ± 2.5 A 50.8 ± 2.6 A	24.1 ± 3.3 A 58.7 ± 1.3 B	30.6 ± 2.8 A 49.3 ± 2.5 A
	Gastric duration
	At 75 rpm 1h I 2h II	18.6 ± 1.1 A,B 24.8 ± 2.5 A	17.4 ± 1.9 A 24.1 ± 3.3 A	19.2 ± 1.5 B 30.6 ± 2.8 A
	At 100 rpm 1h III 2h IV	46.1 ± 1.2 A 50.8 ± 2.6 A	54.6 ± 1.6 B 58.7 ± 1.3 B	50.7 ± 1.2 C 49.3 ± 2.5 A
	Pepsin 1000 U/mL I 2000 U/mL II 4000 U/mL II	26.1 ± 2.1 A 24.8 ± 2.5 A 28.4 ± 0.9 A	15.7 ± 1.7 B 24.1 ± 3.3 A 24.9 ± 1.9 A	19.9 ± 1.1 C 30.6 ± 2.8 A 24.7 ± 1.2 A

Pancreatin At 75 rpm 100 U/mL I 200 U/mL II 400 U/mL III At 100 rpm 100 U/mL IV 200 U/mL V 400 U/mL VI

	24.7 ± 1.6 A	18.4 ± 0.7 B
	24.8 ± 2.5 A	24.1 ± 3.3 A
	34.7 ± 1.6 A	28.8 ± 2.3 B
	45.5 ± 3.6 A
	50.8 ± 2.6 A
	67.7 ± 3.7 A

& considered and termed in the text as marginal digestion conditions

Acknowledgements

The authors are grateful for the support of the Fonds National de la Recherche Luxembourg (Grant No. C16/BM/11320230). The insights obtained from the EU-COST actions EUROCAROTEN (CA15136) and POSITIVE (FA 1403) are much appreciated. The authors thank Yvan Devaux, head of cardiovascular research unit, for placing CVRU facilities at our disposal.


Willy Cei

Harry Archimède

Rémy Arquet

Yoann Felicite

Dalila Feuillet

Angebert Népos

Pierre Mulciba

Tatiana Silou-Etienne

Gisèle Alexandre

Jean-Christophe C Bambou
email: jean-christophe.bambou@inra.fr

W Ceï

& H Archimède

& R Arquet

Y Félicité

& D Feuillet

& A Nepos

Mulciba

& T Etienne

& G Alexandre

Effect of changes in the nutritional status on the performances of growing creole kids during an established nematode parasite infection

Keywords: Nutrition, Parasitism, Small ruminant, Growth performance, Infection

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

Introduction

Gastrointestinal nematode (GIN) infections are among the most common and economically important pathogenic constraints on efficient grazing small ruminant production system [START_REF] Bishop | Possibilities to breed for resistance to nematode parasite infections in small ruminants in tropical production systems[END_REF][START_REF] Charlier | Chasing helminths and their economic impact on farmed ruminants[END_REF]. The rise of anthelmintic resistance during the past decades and public concern about chemical residues in animal products have increased the need for alternative control strategies [START_REF] Jackson | The development of anthelmintic resistance in sheep nematodes[END_REF][START_REF] Papadopoulos | Anthelmintic resistance in sheep nematodes[END_REF]). There is accumulating evidence showing that the nutritional status is closely associated with the capacity of the host to mount an efficient immune response against invading pathogens and more singularly against GIN [START_REF] Adams | Nutrition-based health in animal production[END_REF][START_REF] Colditz | Six costs of immunity to gastrointestinal nematode infections[END_REF]). Thus, nutritional manipulation of small ruminants has long been considered as a tool for the control of GIN infections [START_REF] Hoste | Chapter Seven-Interactions Between Nutrition and Infections with Haemonchus contortus and Related Gastrointestinal Nematodes in Small Ruminants[END_REF]. Numerous feeding trials with small ruminants have paid much attention on the effects of an increased nutrients supply (in protein and/or energy) on the host response to GIN infections [START_REF] Houdijk | Differential effects of protein and energy scarcity on resistance to nematode parasites[END_REF]. It has been shown that an improved nutritional status could reduce the production losses and mortality rates due to GIN infection [START_REF] Athanasiadou | Exploiting synergisms and interactions in the nutritional approaches to parasite control in sheep production systems[END_REF]. Most of the studies investigating the underlying mechanisms show that the nutritional status is closely correlated with the host immune response involved in regulating GIN establishment, fecundity, and survival rates [START_REF] Athanasiadou | Exploiting synergisms and interactions in the nutritional approaches to parasite control in sheep production systems[END_REF][START_REF] Athanasiadou | Dietary Components and Immune Function-Prevention and Treatment of Disease and Cancer[END_REF].

In tropical countries, the variation of feed resources availability and quality lead to seasonal recurrent deteriorated nutritional status [START_REF] Aumont | Sources of variation in nutritive values of tropical forages fron the Caribbean[END_REF][START_REF] Archimede | Tropical resources: availability and feed value[END_REF]. However, in most of the studies, the effect of the nutritional status is evaluated either concomitantly with the infection or before the infection (i.e., the carry-over effect). To our knowledge, the potential beneficial or detrimental effects of nutritional status changes during an establish GIN infection have never been addressed. In this study, we evaluated the effect of changes in the nutritional status on the performances of growing Creole kids during an established experimental Haemonchus contortus infection.

Materials and methods

The experiment was conducted at the Institut National de la Recherche Agronomique (INRA) Plateforme Tropicale d'Expérimentation sur l'Animal (PTEA Gardel), Guadeloupe (16°17′ latitude north, 61°32′ longitude west). All animal care, handling techniques, procedures, as well as license for experimental infection and blood sampling were approved by INRA, according to the certificate number A-971-18-01 of authorization to experiment on living animals issued by the French Ministry of Agriculture, before the initiation of the research.

Animals, management, and experimental design

The study was carried out with growing male 6-month-old Creole kids (n = 18) born and reared outdoors at PTEA Gardel on pastures based on Digitaria decumbens and Brachiaria purpurascens irrigated if needed and fertilized (0.5 kg N/ha/day). Four weeks before the experimental infection, the kids were drenched with ivermectin (Oramec, Merial, Lyon, France, 0.3 mg/kg body weight (BW)), levamisole (Polystrongle, Merial, Lyon, France 7.5 mg/kg BW), and toltrazuril (Baycox Ovis, Bayer HealthCare, Loos, France, 20 mg/kg BW); housed under worm-free conditions in individual pens (2.0 × 1.0 m); and fed with 300 g/kids of a mix of supplements (50:50) of commercial concentrate (CC) and dried banana (Ban). The parasite-free fresh grass (FG) was proposed ad libitum. During this period, the fecal egg counts (FEC) remained at zero (1 FEC/week/animal). The day of the experimental infection, two groups of kids were randomly constituted (infected and non-infected; n = 9 per group; 11.83 ± 2.44 and 12.32 ± 1.88 kg BW, respectively). The experimental infection was performed with an oral single dose of 10 ml of tap water containing 500 third-larva stage (L3)/kg BW of Haemonchus contortus and Trichostrongylus colubriformis (60% H. contortus and 40% T. colubriformis). The L3 were obtained 42 days before the experimental infection from cultures of feces taken from Creole goats at pasture at PTEA Gardel. The non-infected kids were sham infected with 10 ml of tap water. Kids in each group were used in a balanced 2 × 3 factorial split-plot design with three 4-week periods from the experimental infection. Each period of 4 weeks consisted of 2 weeks of adaptation to the diet, followed by 2 weeks of measures (intake, digestibility, FEC, and packed cell volume (PCV)). The experiment lasted for 12 weeks in total. The feed intake, the growth rate (average daily gain (ADG)), the FEC, and the PCV were monitored weekly. The digestibility of the diets was measured during the periods corresponding to 3 and 4, 7 and 8, and 11 and 12 weeks post-infection.

Diets and feedings

The two supplements (CC and Ban; Table 1) were balanced in energy and protein as follow: (1) 12.2 MJ/kg dry matter (DM) and 20.6% of crude protein (CP) (commercial concentrate (CC)), (2) 11.1 MJ/kg DM, and 4.3% CP (dried banana (Ban)). The quantity of supplements (CC and Ban) offered was adjusted on the energy basis to reach the same level of energetic supplementation (270 and 300 g/day/animal for CC and Ban, respectively). The FG was on average at 6.7 MJ/kg DM and 7.9% CP. The supplements (CC or Ban) were distributed at 7 a.m. and the FG at 10 a.m. and 3 p.m. (50% of the daily allowance at each meal). The grass was cut daily 3 cm above the ground with the same mowing machine (BCS S.p.A., Milan, Italy) at 7 a.m. and then chopped into 5-cm lengths using an electric chopper (Dessertine-Hupin S.A., Buxière Les Mines, France).

Feed intake and in vivo total tract digestibility

As previously described, total feces collection was performed with fecal trays placed behind the kids [START_REF] Bambou | Intake and digestibility of naive kids differing in genetic resistance and experimentally parasitized (indoors) with Haemonchus contortus in two successive challenges[END_REF]. Briefly, pooled samples of FG, supplements, and feces (10% per day of the wet weight) were daily collected for chemical analyses. For each digestibility measurement week, a 5-day period for sample collection was carried out. Offered and refused feed were individually recorded weekly in order to determine voluntary dry matter intake (DMI). The in vivo apparent total tract DM, organic matter (OM), CP, neutral detergent fiber (NDF), acid detergent fiber (ADF), and Amidon digestibilities were determined.

Chemical analyses and analytical procedures

The DM contents of feeds, orts, and feces were determined by oven drying (type SE-79, Le Matériel Physic Chimique Flam et Cie, MPC, Neuily S/Marine, France) to a constant weight at 60 °C for 48 h while ash content was determined by heating samples at 550 °C for 4 h; thereafter, the OM content was calculated by difference (AOAC 1997). Dry samples were obtained for further chemical analyses and were ground (model SK100 confort Gußeisen, F. Kurt Retsch GmbH & Co, Haan, Germany) to pass through a 1-mm stainless steel screen. The CP content was calculated after N determination by combustion using the micro-Dumas method (NA2100 Protein, CE Instruments, ThermoQuest S.p.A., Milano, Italy).

The methods of Vansoest et al. (1991) were followed to determine NDF and ADF (sequentially) on an ash-free basis using the ANKOM 200 Fiber Analyzer incubator (ANKOM Technology, Fairport, NY). The hemicellulose and cellulose contents of ingredients were calculated as the differences between NDF and ADF and ADF and ADL, respectively.

Fecal egg count and blood sampling

To determine FEC, fecal samples of approximately 10 g were weekly collected directly from the rectum of each kid. The feces were kept in plastic tubes to avoid contamination and immediately transported to the laboratory in refrigerated vials. All samples were individually analyzed using a modified McMaster method for rapid determination, and FEC was expressed as the number of eggs per gram of feces [START_REF] Aumont | Le dénombrement des éléments parasitaires: Un outil pour l'étude de la résistance génétique aux endo-parasites chez les petits ruminants[END_REF]). Blood samples were individually collected once a week by jugular venipuncture from each Creole kid by using disposable syringes and 20-ga needles. A 2.5-ml portion of each blood sample was placed in commercial anticoagulant tubes (ethylenediaminetetraacetic acid (K 3 EDTA) tubes; Becton Dickinson, Plymouth, UK). Blood samples previously placed in EDTA-coated tubes were used to measure the PCV with the capillary microhematocrit method [START_REF] Goldsmid | Studies on laboratory diagnosis of human filariasis[END_REF].

Calculation and statistical analysis

Data (PCV, FEC, nutrient digestibility, body weight gain, and diet digestibility) collected simultaneously from infected and non-infected animals were analyzed using the MIXED procedure of SAS (v. 9.1, SAS Inst. Inc., Cary, NC, 2003). The FEC were log transformed (Ln (FEC + 15)) in order to normalize the variances. The model included the fixed effects of periods (corresponding to the 2 weeks of measures after adaptation to the diet), parasitological status (PS, infected vs non-infected), diet, and diet × PS, with the effect of animals as random effect. Significance was declared at ≤5% of probability; comparisons between means were tested by the least squares means procedure with adjustment for multiple comparisons (Tukey-Kramer).

Results

The compositions and nutritional values of the supplements and the fresh grass are shown in Table 1. There was a dietary effect on FEC (Table 2, p = 0.022) with the highest value of FEC in the CC diet. No difference was observed between Ban and FG diets for FEC. A significant interaction between PS and diet was observed on PCV, with the lowest PCV in the CC diet of the infected group (Table 2, p = 0.0003). No difference was observed between Ban and FG diets for PCV in the infected group. There was no effect of diet on PCV in the noninfected control groups.

There was a dietary effect on water intake and an interaction between PS and diet on the DMI (Table 3, p < 0.0006). The highest water intake was observed with the CC diet. The DMI decrease significantly in the infected groups whatever the diet. The highest DMI reduction was observed in the FG diet (-66% compared with -47.5 and -51% in the Ban and the CC diets, respectively). The only significant interaction between diet and PS was observed on CP digestibility (Table 3, p = 0.0024). The CP digestibility decreased significantly in the infected groups whatever the diet. The highest reduction was observed in the Ban diet (-59.2% compared with -12.7 and -36.2% in the CC and the FG diets, respectively). No difference between DM, OM, and NDF digestibilities was observed between the Ban and the FG diets, but a reduction was observed in the infected groups whatever the diet (Table 3, p > 0.05). The highest digestibility of DM, OM, and NDF was observed in the CC diet (p < 0.005). The reduction of DM, OM, and NDF digestibilities ranged from -15.6 to -5% with no significant difference between diets. In the non-infected groups, the highest ADG was in the CC diet (p = 0.0001). The ADG of the Ban and the FG diets in the control non-infected groups was low. A significant decrease of the ADG was observed in the infected groups whatever the diet (p = 0.0001). Weight lost was observed in infected groups of the Ban and the FG diet (-4.6 and -25.3 g/day, respectively).

Discussion

The beneficial effect of an improved nutritional status of small ruminants on GIN infection is one of the most easy to implement alternatives to anthelmintics. Numerous studies have investigated the effect of the quality and/or the quantity of feed supplements (in protein and/or energy) on the host response [START_REF] Houdijk | Differential effects of protein and energy scarcity on resistance to nematode parasites[END_REF]Torres-Acosta et al. 2012). According to [START_REF] Houdijk | Differential effects of protein and energy scarcity on resistance to nematode parasites[END_REF], host resistance is mostly affected by metabolizable protein scarcity and moderately to metabolizable energy. Thus, higher protein diets should allow a better control of GIN infection in grazing small ruminants. However, during the last decades, feed costs have tended to increase, affecting the profitability of livestock production, including small ruminants. A step toward recommendation of nutritional strategies allowing a better control of GIN is fine tuning of the feeding programs. Feeding trials with small ruminants have paid much attention on the effect of supplementation on the most susceptible physiological stages (i.e., young animals and periparturient does and ewes) and on the timing of supplementation (concomitant vs carry-over effects) [START_REF] Knox | Nutritional programming of young sheep to improve later-life production and resistance to nematode parasites: a brief review[END_REF][START_REF] Houdijk | Manipulating small ruminant parasite epidemiology through the combination of nutritional strategies[END_REF]. In tropical countries, feed resources are quite variables in quality and in quantity which lead to seasonal deteriorated nutritional status. In this study, we evaluated the short-term impact of changes in the nutritional status on the performances of growing kids during an established GIN infection. The DMI and digestibility increased significantly with the CC diet whatever the parasitological status (i.e., infected vs non-infected). The crude protein digestibility was lower in the infected groups but higher with the CC diet whatever the parasitological status. The NDF digestibility was also higher with the CC diet compared with the FG and the Ban diets. The lower digestibility of CP in infected animals is likely related with the already known hematophagous activity of both larval and adult stages of H. contortus and the subsequent mucosal hemorrhages coupled with the modifications of nitrogen metabolism induced by T. colubriformis (Williams and Palmer 2012). A poor reutilization of the absorbed nitrogen for de novo protein synthesis has been suggested, since a higher level of non-urea nitrogen excretion was measured in GIN-infected sheep (Williams and Palmer 2012). As many components of the immune system, such as immunoglobulins, muco-proteins, and cellular products such as leukotriennes, are proteinaceous, a decrease availability of amino acids for the metabolism would result to a decrease capacity to mount and maintain a protective immune response [START_REF] Coop | Nutrition-parasite interaction[END_REF]). Indeed, it has been previously showed, by comparing groups of animals fed with different diets, that the prolificacy of GIN decreases with an improved nutritional status in both sheep and goats [START_REF] Hoste | Chapter Seven-Interactions Between Nutrition and Infections with Haemonchus contortus and Related Gastrointestinal Nematodes in Small Ruminants[END_REF]. We showed a significant effect of the diet changes on the FEC. Surprisingly, here, we showed that the diet with higher protein contents increased the intensity of the GIN infection measured trough a significant increase of the FEC correlated with a decrease of the PCV. However, in contrast with the lower nutritional status (i.e., FG and Ban diets), this diet maintained higher feed intake and digestibility and growth rate. In growing animals, a period of nutritional restriction induces a compensatory growth phenomenon which is the result of the optimization of different physiological functions of the animal with the objective to increase the growth rate [START_REF] Hoch | Mechanisms and consequences of compensatory growth in ruminants[END_REF]. In this study, the higher growth rate observed for the CC diets with a higher protein nutritional status could be the result of the poor protein nutritional status of the FG and the Ban diets which would have effects similar to a nutritional restriction.

Numerous studies suggest that response to an immunological challenge must be traded off against other physiological functions including growth [START_REF] Shudo | Inducible defense against pathogens and parasites: Optimal choice among multiple options[END_REF]van der Most et al. 2011). Interestingly, the results of the present study suggest that the nutritional status could affect these physiological trade-offs between the growth and the immune response against GIN infection. The improvement of the nutritional status after a period with a deteriorated nutritional status during an establish GIN infection would improve the animal performance at the expense of the mechanism involved in the control of the infection. This finding could be of interest in tropical countries, where the deleterious effect of an establish GIN infection potentiated by seasonal deteriorated nutritional status could be counterbalanced by a protein-rich diet.

2012. Nutritional manipulation of sheep and goats for the control of gastrointestinal nematodes under hot humid and subhumid tropical conditions, Small Ruminant Research, 103, 28-40 van der Most, P.J., de Jong, B., Parmentier, H. K., Verhulst, S., 2011. Trade-off between growth and immune function: a meta-analysis of selection experiments, Functional Ecology, 25, 74-80

Vansoest, P.J., Robertson, J.B., and Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition, Journal of Dairy Science, 74, 3583-3597 Williams, A.R., and Palmer, D.G., 2012. Interactions between gastrointestinal nematode parasites and diarrhoea in sheep: Pathogenesis and control,Veterinary Journal,192,[279][280][281][282][283][284][285]

Table 1

 1 Chemical composition and nutritional values of the supplements and the fresh grass FG fresh grass, OM organic matter, CP crude protein, NDF neutral detergent fiber, ADF acid detergent fiber, ADL acid detergent lignin, ME metabolizable energy

	Item %	CC	Dried banana	FG
	DM	92.0	83.4	28.3
	OM	85.0	94.3	87.9
	CP	20.6	4.3	7.9
	NDF	18.2	15.3	67.7
	ADF	4.0	8.0	37.2
	ADL	0.4	3.7	4.6
	Amidon	37.2	70.7	0
	ME (MJ/kg DM)	12.2	11.1	6.7
	CC commercial concentrate (ingredients: soybean meal, wheat bran,
	maize),

  CC commercial concentrate, fresh grass supplemented with commercial concentrate, Ban dried banana, fresh grass supplemented with dried banana, FG fresh grass non-supplemented, I infected, period after experimental infection, NI non-infected, period non-infected, PS parasitism status, infected versus non-infected period, DMI dry matter intake, DM dry matter, OM organic matter, CP crude protein, NDF neutral detergent fiber, ADF acid detergent fiber, ADG average daily gain CC commercial concentrate, fresh grass supplemented with commercial concentrate, Ban dried banana, fresh grass supplemented with dried banana, FG fresh grass non-supplemented, I infected, period after experimental infection, NI non-infected, period non-infected, PS parasitism status, infected versus non-infected period, FEC fecal egg counts, eggs per gram of feces, PCV packed cell volume

	Table 2 Least squares means of
	FEC and PCV of Creole kids		CC		Ban			FG			p value
	infected with an oral single dose
	of 500 third-larva stage (L3)/kg		I	NI	I		NI	I	NI	SEM	Diet		PS	Diet × PS
	BW of Haemonchus contortus
	and Trichostrongylus	FEC	3783a	-	3302b	-	3128b	-	750	0.022	-	-
	colubriformis or not and feed with	PCV	19a	26b	22c		26b	22c	29d	2.0	0.0001	0.0001	0.0003
	three different diets
		Univariate analysis of FEC and PCV data. LS means with different lowercase letters within lines are significantly
		different (p < 0.05)
	Table 3 Least squares means of
	water intake, DMI, and apparent	Item		CC		Ban			FG			p value
	total tract digestibility of Creole
	kids infected with an oral single			I	NI	I		NI	I	NI	SEM Diet	PS	Diet ×
	dose of 500 third-larva stage (L3)/												PS
	kg BW of Haemonchus contortus
	and Trichostrongylus	Water intake	943a 949a	586b	736b	541b	652b 54.0	0.0001 0.3021 0.6011
	colubriformis or not and feed with	(ml/day)
	three different diets	DMI (g/day) 354a 717b	301a	573c	245d	553c 58.5	0.0001 0.0001 0.0055
		Digestibility (g/kg of)
		DM		723a 782b	598c	709a	583d	661a 13	0.0001 0.0001 0.23
		OM		727a 784b	635c	708d	621c	654d 21	0.0001 0.0035 0.45
		CP		660a 756b	209c	512d	371e	582d 38	0.0001 0.0001 0.0024
		NDF		600a 674b	546c	625d	578c	653d 26	0.0365 0.0013 0.99
		ADF		479a 634b	507c	574d	529c,d 615b 40	0.5028 0.0013 0.216
		Amidon		999a 996a	979b	991b,a -	-	5.0	0.0310 0.5004 0.2355
		ADG		9.7a	107.9b -4.6c 1.3d	-25.3e 7.4a	6.0	0.0001 0.0001 0.0001
		(g/day)
		Univariate analysis of water intake, DMI, and apparent total tract digestibility data. LS means with different
		lowercase letters within lines are significantly different (p < 0.05)

Acknowledgments The authors are grateful to the Gardel team in charge of the goat flock: R. Arquet, T. Kandassamy, W. Troupé, J. Gobardhan, and S.-A. Matou. This study was funded by the Project Agroecodiv (La Région Guadeloupe and Fonds Européens FEDER). WC was supported by a doctoral fellowship from la Région Guadeloupe and the animal Genetics Department of INRA.

Compliance with ethical standards

Statement of animal rights All the investigations presented in this study were approved by a local ethic committee (CEMEAAG, Comité d'Ethique en Matière d'Expérimentation Animale des Antilles et de la Guyane) and the French Ministry of Higher Education and Research.

Conflict of interest

The authors declare that they have no conflict of interest.