Roua Lajnaf
email: roua_lajnaf@yahoo.fr

Ahmed Zouari

Ines Trigui

Hamadi Attia

M A Ayadi

Effect of different heating temperatures on foaming properties of camel milk proteins: A comparison with bovine milk proteins

The effect of different heat-treatment temperatures (70, 80, 90 and 100 °C) on the foaming properties (foam capacity and stability) and the physicochemical characteristics (surface hydrophobicity, ζ-potential, and interfacial tension values) of camel milk proteins was investigated. Overall, the results showed that while increasing the temperature, greater foamability was measured for camel milk proteins (up to 165%). This behaviour was linked to the heat denaturation and aggregation of camel milk proteins, which led to an increase in the surface hydrophobicity and a decrease in the electronegative charge and interfacial tension. Likewise, our results indicated that the highest β-casein amount in camel milk (~44% of total proteins) as well as its secondary structure (β-sheet conformation) and its high hydrophobicity, regulated the foaming mechanism of camel milk proteins.

Introduction

Known in the arid countries as a culturally important drink, camel milk is gaining popularity internationally due to its claimed biological values and its therapeutic properties such as anti-diabetic [START_REF] Mudgil | Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates[END_REF][START_REF] Nongonierma | Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of camel milk protein hydrolysates generated with trypsin[END_REF], 2018), hypo-allergic and anti-cancer properties [START_REF] Kamal | Inhibitory properties of camel whey protein hydrolysates toward liver cancer cells, dipeptidyl peptidase-IV, and inflammation[END_REF]. Consequently, the production of this milk has substantially increased on large commercial scales from modern farms [START_REF] El-Agamy | Are camel milk proteins convenient to the nutrition of cow milk allergic children?[END_REF].

Camel milk is different from cow milk in its protein composition and the structure of its proteins, leading to different techno-functional and biological properties [START_REF] Hailu | Functional and technological properties of camel milk proteins: a review[END_REF][START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF]. As for milk of other milk-producing mammalian species, camel milk can be classified into two major components of proteins, i.e., caseins and whey proteins.

Caseins constitute the main protein fraction of the camel milk representing 52-87% (w/w) of the total milk proteins. β-Casein is the most abundant casein in camel milk followed by αS1casein, constituting about 65% and 22% of the total caseins content, respectively, compared with 39% and 38% in bovine caseins, respectively. Only 3.3% of the total camel milk casein corresponds to κ-casein, compared with bovine κ-casein being 13% of the total casein in cow milk [START_REF] El-Agamy | Camel milk[END_REF][START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF][START_REF] Hailu | Functional and technological properties of camel milk proteins: a review[END_REF][START_REF] Kappeler | Sequence analysis of Camelus dromedarius milk caseins[END_REF].

Whey proteins are the second main protein fraction of camel milk, constituting 24.5% (w/w) of the total proteins [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF]. However, whilst in cow milk β-lactoglobulin (β-Lg) is the main component of the whey proteins (72%) followed by α-lactalbumin (α-La) (24%), β-Lg is totally absent in camel milk [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF][START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF][START_REF] Maqsood | Comparative characterization of protein and lipid fractions from camel and cow milk, their functionality, antioxidant and antihypertensive properties upon simulated gastrointestinal digestion[END_REF][START_REF] Merin | Short communication A comparative study of milk serum proteins in camel (Camelus dromedarius) and bovine colostrum[END_REF][START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF].

Thus, the α-La is the major camel whey protein with an average concentration of 2.3 g L -1 , a significantly higher value than that for its bovine counterpart (1.1 g L -1 ; [START_REF] Chatterton | Bioactivity of βlactoglobulin and α-lactalbumin -Technological implications for processing[END_REF][START_REF] El-Hatmi | Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum[END_REF].

Milk foams are defined as colloidal systems where the air-bubbles created are stabilised by a matrix composed of surface-active agents of milk that are mainly proteins [START_REF] Dickinson | Hydrocolloids at interfaces and the influence on the properties of dispersed systems[END_REF]. As for other techno-functional properties of proteins, foaming behaviour is determined by the type of protein and environmental factors including ionic strength, pH and the heat-treatment temperature [START_REF] Lorenzen | Verfahren zur modifizierung von Milcheiweiß[END_REF]. For bovine milk, the thermal denaturation of proteins has been reported as the elementary step of the reactions leading to the heat-induced aggregation of disulphide-linked proteins. Thus, under a heat-treatment of 60 °C, β-Lg dissociates from a dimeric structure to native monomers. The thermal denaturation of this protein leads to thiol-disulphide exchange reactions and thus heatinduced association of whey proteins and their aggregates with caseins [START_REF] Kazmierski | Characterization of soluble aggregates from whey protein isolate[END_REF][START_REF] Roefs | A model for the denaturation and aggregation of β-lactoglobulin[END_REF]. Therefore, the resulting foaming properties will be a complex competitive adsorption phenomena between proteins in their native, nonaggregated denatured, and aggregated states [START_REF] Schmitt | Whey protein soluble aggregates from heating with NaCl: physicochemical, interfacial, and foaming properties[END_REF].

The foaming properties of bovine milk have been widely studied and reported since the beginning of the last century [START_REF] Borcherding | Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk[END_REF][START_REF] Hatakeyama | Effects of manufacturing conditions on the foaming properties of milk and sensory characteristics of foamed milk[END_REF][START_REF] Kamath | The influence of temperature on the foaming of milk[END_REF]. For camel milk, previous studies noted that that it is greatly frothy, even if when it is shaken slightly [START_REF] Shalash | The production and utilization of camel milk[END_REF], but little research exists on the foaming properties of camel milk proteins (Al-Shamsi, Mudgil, Hassan, & Maqsood, 2018) and their interfacial properties have not been thoroughly described in the literature. Thus, different behaviours can be suggested for the camel milk, as the different protein composition would have a great impact on the resulting foaming properties of milk after a heat-treatment.

An investigation of the foaming and interfacial behaviour of camel milk at different heating temperatures compared with that of bovine milk could be a very useful tool to control and predict the functionality of dairy systems in both milks. Despite its high production and consumption, cow milk is associated to different nutritional problems, such as allergy. Thus, camel milk was recently suggested as a food alternative to cow milk. Aerated dairy products of camel milk could be of great interest to foam industry. Indeed, fresh camel milk and its products have unique flavour and good nutritional and therapeutic properties such as anticarcinogenic, anti-diabetic, and anti-hypertensive properties [START_REF] Al Haj | Compositional, technological and nutritional aspects of dromedary camel milk[END_REF]. Furthermore, camel milk has been recommended to be consumed by children who are allergic to bovine milk as reported by [START_REF] El-Agamy | Are camel milk proteins convenient to the nutrition of cow milk allergic children?[END_REF].

Camel milk aerated products can compete in the market with those of cow milk as it could have the same attractive textural characteristics of milk foams with important nutritional value, especially to patients allergic to cow proteins. Starting from these considerations, the aim of this work was to investigate the foaming and interfacial properties of bovine and camel milk after different heating temperatures for potential applications of camel milk in the industrial foam production, which could be of great importance to dairy industry.

Materials and methods

Milk samples

Fresh camel milk was collected from 20 different healthy female camels of the same breed (Camelus dromedarius), ranging between 2 and 12 months in lactation stage, and belonging to a farm located in the south of Tunisia. Fresh cow milk was derived from a local breed located in the region of Sfax in Tunisia.

Both milk samples were directly transported to the laboratory using cooler bags (at 4 °C). They were then systematically skimmed by centrifugation (Thermo Scientific Heraeus Megafuge centrifuge, Germany) at 3000 × g for 20 min at 4 °C (Felfoul, Lopez, Gaucheron, Attia, & Ayadi, 2015a). Both milk types samples were then freeze dried (Bioblock Scientific

Christ ALPHA 1-2, IllKrich-Cedex, France) to obtain powders and stored at -20 °C for further use.

Heat-treatment experiments

Lyophilised milk samples were dissolved in deionised water (Milli-Q system, Millipore, USA) at a level of 1 g L -1 . Camel and bovine milk (1g L -1 of protein concentration)

were heated using water bath at 70 °C, 80 °C 90 °C and 100 °C for 30 min as reported in previous studies (Felfoul, Lopez, Gaucheron, Attia, & Ayadi, 2015b;[START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF].

After heating, beakers containing milk camel and bovine milk were put on ice to stop milk protein denaturation. The control milk sample was at 20 °C corresponding to native conditions without heating [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF][START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF].

Foaming properties

Ten millilitres of camel and bovine milk at a protein concentration of 1 g L -1 were poured in a glass measuring cylinder (radius 1.5 cm × length 7.5 cm) and whipped using an appropriate mixer (Ultra Turrax T25, IKA Labortechnik, Staufen Germany) at 13,500 rpm for 2 min at room temperature (~25 °C) [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF][START_REF] Marinova | Physico-chemical factors controlling the foamability and foam stability of milk proteins: Sodium caseinate and whey protein concentrates[END_REF]. Immediately after whipping, the volume of the created foam was read from the cylinder. Afterwards, foam capacity (FC) was calculated using equation Eq. ( 1):

FC = (VF /V0) × 100 (1)
where VF is the volume of the created foam and V0 the volume of the initial milk before mixing.

The time for the draining of the volume of foam to half was also measured to determine the foam stability (FS) as described by [START_REF] Marinova | Physico-chemical factors controlling the foamability and foam stability of milk proteins: Sodium caseinate and whey protein concentrates[END_REF].

Purification and structural characteristics of camel β-casein

Purification of camel β-casein

The bovine and camel β-caseins were extracted using the technique described by [START_REF] Huppertz | A method for the large-scale isolation of β-casein[END_REF] and modified by [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF].

After defatting camel milk, casein fraction was separated from the whey by rennet coagulation in the presence of 1.4 mL rennet enzyme per litre of camel milk (Mucor miehei, strength = 1:10,000, Laboratories Arrazi, Parachimic,Sfax, Tunisia) at 37 °C for 1-2 h followed by centrifugation at 5000 × g for 20 min at 20 °C (Fig. 1). The rennet content added was four times higher in camel milk than for bovine milk (0.35 mL L -1 ), as reported in previous works (Felfoul et al., 2015b;[START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF][START_REF] Lajnaf | Comparative study on emulsifying and physico-chemical properties of bovine and camel acid and sweet wheys[END_REF][START_REF] Ramet | The technology of making cheese from camel milk (Camelus dromedarius). Rome, Italy: Food and Agriculture Organisation of the United Nations[END_REF]. This behaviour was attributed to the differences in the size of casein particles due to reduced κ-casein content [START_REF] Al Haj | Compositional, technological and nutritional aspects of dromedary camel milk[END_REF].

Afterwards, a volume of preheated demineralised water (80 °C) equal to that of the discarded whey was added to the curd and the mixture was kept at 80 °C for 5 min to disable the action of rennet enzyme, then centrifuged for 5000 × g for 15 min at 20 °C. After discarding the supernatant, the curd was kept, macerated and suspended in demineralised water (5 °C) (volume equal to that of the discarded whey previously). Finally, the protein suspension was kept at 5 °C for up to 24 h and centrifuged at 5000 × g for 15 min at 5 °C.

The supernatants obtained after centrifugation containing the camel and bovine β-caseins were kept at -18 °C then lyophilised (Bioblock Scientific Christ ALPHA 1-2) for further analysis.

The purified freeze dried camel β-casein was used without further modifications or heating treatment.

Infra-red spectroscopic analysis

The absorption spectra of the native lyophilised camel and bovine β-caseins were obtained by FT-IR spectroscopy (Perkin Elmer®, Spectrum™ 100, Singapore) equipped with attenuated total reflection (ATR) accessory containing a diamond/ZnSe crystal. The FT-IR spectra were recorded in the 4000 and 600 cm -1 range at room temperature.

Nuclear magnetic resonance analysis

1 H nuclear magnetic resonance (NMR) analysis of purified camel and bovine βcaseins in their native state was carried out according to the method of Fernández et al.

(2012) with some modifications. Twenty milligrams of lyophilised β-casein sample was dissolved in 500 µL D2O and put into NMR tube. One-dimensional 1 H NMR spectra were recorded at 400 MHz and 25 °C on a Bruker 600M spectrometer (Rheinstetten, Germany).

High performance liquid chromatography analysis

The effect of the different heat-treatments (70, 80, 90, and 100 °C for 30 min) on camel milk proteins was examined by high performance liquid chromatography (HPLC;

Agilent 1260 Infinity quaternary LC, Germany) using the method of [START_REF] Yüksel | Detection of the milk proteins by RP-HPLC[END_REF]. Milk proteins were separated on a C18 column RP-HPLC column (Zorbax Eclipse Plus C18, 250 mm length × 4.6 mm, particle size 5 µm, Packing Lot: B14292) and then analysed using a Shimadzu SPD6A-UV detector measuring the optical density at 220 nm for 40 min.

Overall, 500 μL of milk sample were added to 3.7 mL of a solution containing solvent A (acetonitrile, water, and trifluoroacetic acid, 100:900:1, by vol) and solvent B (acetonitrile, water, and trifluoroacetic acid in a ratio 900:100:1 by vol) in a 70:30 ratio (v/v) [START_REF] Jafar | Camel whey protein hydrolysates displayed enhanced cholesteryl esterase and lipase inhibitory, antihypertensive and anti-haemolytic properties[END_REF]. The mixture was vortexed for 10 s and then filtered through 0.45 µm nylon filter before injection into the column with a volume of 20 µL.

When the sample was injected, a gradient was generated immediately after sample injection by increasing linearly the proportion of solvent B as function of time from 20% to 46% at the end of the run (30-40 min). The column temperature was maintained at 25 °C and the mobile flow rate was fixed at 1.0 mL min -1 .

Standard individual bovine proteins (β-casein, αS-casein, κ-casein, β-Lg and α-La)

were purchased from Sigma Aldrich. Individual standards were diluted in solvent A and solvent B mixture (70:30, v/v), separately. For camel milk, chromatograms of camel casein fraction and whey were used for the determination of each camel protein of camel milk.

Thus, 500 μL of camel milk caseins and whey fractions were added to 3.7 mL of the solution containing solvent A and solvent B mixture (70:30, v/v) as milk and bovine standards, separately. Furthermore, a quantitative estimation of each camel milk proteins percentages and the comparison of the data with those of previous works was used to confirm the camel milk composition [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Hailu | Functional and technological properties of camel milk proteins: a review[END_REF][START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF]. Quantitative estimation of major camel and bovine milk protein percentages was performed by calculating the area of the peak of each protein.

Thiol group concentration and denaturation rate

The free thiol group concentration was determined as described by [START_REF] Ellman | Tissue sulfhydryl groups[END_REF].

First, 300 µL of camel and bovine milk at a protein concentration of 1g L -1 were mixed with 50 µL 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) solution (consisting of 2 mM DTNB and 50 mM sodium acetate in deionised water), 100 µL 1 M Tris, pH 8.0, and 550 μL deionised water. The mixture was then incubated at 37 °C for 5 min and finally the optical density was measured at 412 nm using a UVmini-1240 spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

The concentration of thiols in milk (CSH, M) was calculated using Eq. ( 2):

CSH = (OD412nm/ ε412) × (1000/300) (2)
where OD412nm is the absorbance at 412 nm; ε412 is the molar extinction coefficient of the DTNB (13,600 M -1 cm -1 ) at 412 nm, 300 µL is milk volume (at a protein concentration of 1 g L -1 ) and 1000 µL is the total volume of the cuvette.

After determining the concentration of thiols in milk (CSH), the milk protein denaturation rate (DR) values were calculated using Eq. ( 3):

DR (%) = [CSH (heated milk) -CSH (native milk)] / CSH (native milk) × 100 (3)

SDS-PAGE

SDS-PAGE (15% acrylamide gel) experiments were carried out using the technique described by [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] and [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF]. Lyophilised milk samples were dissolved in deionised water at a concentration of 7 g L -1 . After applying heat treatment (70, 80, 90 or 100 °C for 30 min) at this protein concentration, 5 µL of this protein solution (containing 35 µg protein) were taken and added to 5 μL sample buffer [0.5 M Tris-HCl pH 6.8, 10% (w/w) SDS, 2% (w/w) glycerol, 0.5 M β-mercaptoethanol, 0.1% (w/w) bromophenol blue] and heated at 95 °C for 5 min. Electrophoresis was performed at constant current 120 V for 2 h (Mini Protean Tetra Cell, BioRad laboratories, USA) and the gel was stained for 12 min with 2.5% (w/v) Coomassie blue R-250 in a mixture of 50% ethanol and 10% acetic acid (v/v). Then, the gel was detained in water solution containing 10% ethanol and 14% citric acid. A mixture of the pre-stained marker proteins (14.4-94 kDa) was subjected to the same procedure described above. The molecular masses of the different milk proteins were estimated by comparing their electrophoretic mobilities with those of marker proteins.

ζ-potential measurements

The ζ-potential values of camel and bovine milk at a protein concentration of 0.5 g L -1

were determined at 25 ± 1 °C using the Zetasizer Nano-ZS90 apparatus (Malvern Instruments, Westborough, MA) as suggested by [START_REF] Magnusson | Interactions between hydrophobically modified starch and egg yolk proteins in solution and emulsions[END_REF]. The ζpotential value (mV) was determined using Henry's equation (Eq. 4):

UE = (2εζf(kα))/3ɳ ( 4
)
where UE is the electrophoretic mobility, f(kα) is the function related to the ratio of particle radius (α, nm) and the Debye length (k, nm -1 ), ε is the permittivity (Farad m -1 ) and ɳ is the dispersion viscosity (mPa s) [START_REF] Mcclements | Food emulsions: principles, practices, and techniques[END_REF].

2.9.

Determination of protein hydrophobicity

Hydrophobicity for each milk protein solution was determined by the method described by [START_REF] Chelh | A simplified procedure for myofibril hydrophobicity determination[END_REF] and [START_REF] Al-Shamsi | Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems[END_REF].

Briefly, 1 mL of milk sample (at a protein concentration of 1 g L -1 ) and 200 µL of 1 mg mL -1 bromophenol blue (BPB) solution in distilled water were added and mixed well.

A control consisted of the addition of 200 µL of BPB solution (1 g L -1 ) to 1 mL 20 mM Tris-HCl buffer, pH 8.0, instead of milk sample.

Milk samples and controls were kept under agitation during 10min at room temperature and then centrifuged at 2000 × g at 25 °C for 15min (Thermo Scientific Heraeus Megafuge). Supernatants were diluted 1:10 with distilled water and the optical density was measured at 595 nm using UVmini-1240 spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

The amount of bound-BPB molecules was calculated using Eq. ( 5):

Bound-BPB (µg) = [(OD595 (control) -OD 595 (sample)]/OD 595 (control) × 200 µg (5)

Interfacial tension

The interfacial tension for each camel and bovine milk at a protein concentration of 1 g L -1 was measured using a TSD 971 Tensiometer (Tensiometry System Digital Gibertini Elettronica, Italia) using The Du Noüy methodology as described by Lam and Nickerson (2015a). Thus, for the determination of the interfacial tension of camel and bovine milk at the air-water interface, 20 mL of 1 g L -1 milk protein solution were added within a 40 mm diameter glass sample beaker, followed by the immersion of the Du Nüoy ring (20 mm diameter). Finally, the ring was pulled upwards to stretch the air-water interface of the protein solution to measure the maximum force (Fmax) and then to calculate the interfacial tension value (γ) using Eq. ( 6)

γ = Fmax/(4πRβ) (6)
where γ is the interfacial tension (mN m -1 ), R is the radius of the used ring (20 mm), Fmax is the maximum force (mN), β is a correction factor which depends on two main factors: the dimensions of the ring and the density of the liquid. All interfacial tension measurements (γ)

were carried out at 25 °C.

Statistics

All measurements of each experiment in this work were performed at least in triplicate and mentioned as the mean value ± one standard deviation. Analysis of variance, ANOVA, was performed to test for significance in the main effects of the milk (camel and bovine milk) and heat-treatment conditions (70 °C, 80 °C, 90 °C and 100 °C), along with their associated interactions on the foaming properties and physico-chemical characteristics of milk (surface tension measurements, ζ-potential, DR and hydrophobicity). Statistical analyses were carried out using an appropriate software (IBM SPSS statistics, Version 19, IBM SPSS, USA).

Results and discussion

Foaming properties of milk

Fig. 2 shows the average values of foam capacity (FC) and foam stability (FS) respectively of camel and bovine skim milk as function of the temperature of the heat-treatment. The same milk protein concentration (1 g L -1 ) was chosen for both milk samples to make a significant comparison of foaming properties under native conditions (20 °C) and after heating (70, 80, 90 and 100 °C for 30 min).

Fig. 2a shows that camel milk gives better foam than bovine milk regardless of heating temperature value. Under native conditions, i.e., at 20 °C, camel milk was found to

give better FC values (~138%) than that of bovine milk (~115%) suggesting that in these conditions, camel milk proteins coat the air bubbles better than bovine milk proteins. This behaviour can be explained by greater amount of β-casein in camel milk when compared with bovine milk as reported by [START_REF] Kappeler | 5′-Flanking regions of camel milk genes are highly similar to homologue regions of other species and can be divided into two distinct groups[END_REF].

These results are in agreement with those of [START_REF] Brooker | The development of structure in whipped cream[END_REF] and [START_REF] Ward | EDTA-induced dissociation of casein micelles and its effect on foaming properties of milk[END_REF] who reported that the foamability of milk increased with an increasing content of the β-casein up to a certain degree and then stayed constant. Indeed, this protein is considered as the most surface-active milk protein due to its particular unordered structure and its high hydrophobicity. Thus, the β-casein is the first adsorbed and predominant protein at the air-water interface when compared with other milk proteins [START_REF] Dickinson | Proteins at interfaces and in emulsions. Stability, rheology and interactions[END_REF][START_REF] Zhang | Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins[END_REF]. For camel milk, β-casein was found to play the main role in the creation of camel milk foams; the foamability increased with the β-casein amount in bovine and camel proteins mixtures [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF].

Fig. 2a also shows that, for bovine milk, a thermal treatment during 30min at 70 °C, 80 °C or 90 °C induced a significant increase of the FC from 140% to 154% and then to 169% (P < 0.05), respectively. No significant increase was observed in FC values between 90 °C and 100 °C. For the camel milk, statistical analysis showed that heating improved significantly the foamability (P < 0.05) in comparison with that of native milk, with better foaming achieved after a heat-treatment at 90 °C and 100 °C. However, no significant differences in the FC values (165%) could be found when heat-treatment was applied at 70 °C and 80 °C.

These results are in agreement with those reported by [START_REF] Kamath | The influence of temperature on the foaming of milk[END_REF] in a study carried out with skim and whole bovine milk. Indeed, the high foam of heated milk at temperature values up to 85 °C is mainly due to the decrease in viscosity of heated milk with increasing temperature leading consequently to a faster adsorption of proteins on the interface. Heated milk has better foaming properties due to the partial unfolding state of globular proteins after heating, leading to the exposure of their reactive functional groups.

Therefore, the overall hydrophobicity and flexibility of milk proteins increased resulting a higher foam volume [START_REF] Bals | Effect of pre-heating on the foaming properties of whey protein isolate using a membrane foaming apparatus[END_REF][START_REF] Borcherding | Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk[END_REF][START_REF] Graham | Proteins at liquid interfaces: I. Kinetics of adsorption and surface denaturation[END_REF].

Fig. 2a shows also that heating camel and bovine milk at 100 °C significantly reduced their foamability (P < 0.05) in comparison with the FC values of 90 °C with a better foaming achieved with the camel milk (195%). These results are in agreement with those of [START_REF] Lam | The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate[END_REF] who reported that a higher heating temperature of milk proteins at neutral pH level leads to greater surface hydrophobicity caused by protein unravelling. This behaviour could explain the reduction of the ability of whey proteins to create emulsions [START_REF] Lam | The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate[END_REF].

In the case of camel milk, Lajnaf, Picart-Palmade, Attia, Marchesseau, and Ayadi (2017) found that a thermal treatment at 70 °C or 90 °C for 30min induced a significant increase of the foam volume of the purified camel α-La solutions. This behaviour was explained by the charge repulsion forces of the camel α-La molecules leading to a better adsorption at the air-water interface, which preserve this protein from thermal aggregation after heating.

Fig. 2b shows the foaming stability (FS), i.e., the half-time of liquid drainage from the foams created by bovine and camel milk (at a concentration of 1 g L -1 ) in response to thermal treatment. First, foams made with bovine milk proteins were found to be more stable than those made with camel milk regardless of heating temperature. This behaviour can be explained by the highest amount of the κ-casein in bovine milk [START_REF] El-Agamy | Camel milk[END_REF][START_REF] Kappeler | Sequence analysis of Camelus dromedarius milk caseins[END_REF]. [START_REF] Closs | Effect of chemical glycosylation on the surface properties of the soluble fraction of casein[END_REF] reported that the stability of milk foams is determined by κcasein predominantly due to its particular structured form as compared with other caseins.

Furthermore, [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF] found that bovine β-casein is characterised by a higher ability to stabilise foams when compared with its camel counterpart due to the difference in their molecular structures. β-Lg could also play an important role in the stability of bovine milk. Indeed, the stability of β-Lg foams is higher than those obtained with both camel and bovine α-La [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF].

The stability of foam formed from camel and bovine milk increased significantly with increasing preheating temperature up to 90 °C (P <0.05), above which less stable foams were formed (at 100 °C). For bovine milk, an increase of heating temperature from 20 °C to 90 °C, greatly increased the FS value to 720 s, which represents an increase of 600 s (Fig. 2b). For camel milk, the FS values increased significantly from 60 s to 690 s in this temperature range (P < 0.05). This is in agreement with [START_REF] Dickinson | Hydrocolloids at interfaces and the influence on the properties of dispersed systems[END_REF] and [START_REF] Borcherding | Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk[END_REF] who reported that heating induces an increase in stability of milk foam due to an increase in the adsorption velocity and the diffusion of heated milk proteins at the interface and a decrease in the apparent viscosity of milk. On the other hand, [START_REF] Tosi | Foaming properties of sweet whey solutions as modified by thermal treatment[END_REF] observed that heating could significantly improve the stability of foams formed by sweet whey proteins (2-4 times more stable) when compared with native whey. However, temperature must not be higher than 85 °C for 750 s to avoid an excessive denaturation of proteins reducing their ability to stabilise foam.

For camel milk, [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF] reported that the stability of foam formed by the acid camel whey greatly increased; they found that the acid camel whey presented better properties than bovine whey to create and stabilise foams, with a significant increase of these properties after heating (at 70 °C and 90 °C for 30 min) due the extensive aggregation of the α-La. Indeed, the α-La aggregates contributed to improve the foam stability of camel whey solutions [START_REF] Lajnaf | The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk[END_REF].

Structural characteristics of camel milk protein: β-casein

Infra-red spectroscopic analysis

Fig. 3a shows the FT-IR spectrum of the native β-caseins obtained from camel and bovine milk without further modification. If the protein fractions obtained of both β-caseins are compared, it is evident that both proteins have a very similar spectrum. Such behaviour can be explained by the sequence similarity and identity between these two proteins which are 84.5% and 67.2%, respectively [START_REF] Barzegar | Chaperone activities of bovine and camel β-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation[END_REF].

Both β-casein sample spectra showed the same bands in the region of 3400-3100 cm - 1 , which corresponded to the N-H stretch of amide A and primary amine. Furthermore, the peaks in the region of 2800-2960 cm -1 corresponded to the stretching vibration of C-H which are mainly found in aliphatic side chain of proteins [START_REF] Santoni | Evaluation of alternative vegetable proteins as wood adhesives[END_REF][START_REF] Siu | Physicochemical and structural properties of oat globulin polymers formed by a microbial transglutaminase[END_REF]. The amide I band (1600-1700 cm -1 ), which is the band of the C=O stretching vibrations, is the most important vibrational bands of the protein skeleton. Indeed, this band was associated with the secondary structure of β-casein as reported by [START_REF] Cao | Elucidating the interaction mechanism of eriocitrin with β-casein by multi-spectroscopic and molecular simulation methods[END_REF].

The peak positions of amide I bands were 1642 cm -1 and 1627 cm -1 for camel and bovine β-caseins, respectively, indicating different secondary structure of both β-caseins, especially in the β-sheet structure in accordance with [START_REF] Cao | Elucidating the interaction mechanism of eriocitrin with β-casein by multi-spectroscopic and molecular simulation methods[END_REF]. Indeed, the amide I band is the most sensitive spectral region of the secondary structure of protein. The amide I band frequency assignments for secondary structures are especially α-helix (1654-1658 cm -1 ) and β-sheet (1624-1642 cm -1 ) [START_REF] Kong | Fourier transform infrared spectroscopic analysis of protein secondary structures[END_REF]. This result could explain the higher foam stability of bovine milk when compared with camel milk as the β-casein plays the main role in stabilising milk foams [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF].

NMR spectroscopy data

The representative 1 H NMR spectra obtained from the native purified camel and bovine β-caseins without further modification are illustrated in Fig. 3b. The 1 H NMR spectra showed the sharp proton resonance and the chemical shifts variations of protein in the range of 0-1 ppm. In the spectrum of both proteins, methyl signals are observed in the range 0.7 to 0.9 ppm. The intensity of these signals was more pronounced for camel β-casein when compared with its bovine counterpart. Indeed, this is the region where most amino acids containing methyl groups are, and in particular those in random coil regions of the protein [START_REF] Fernández | Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin[END_REF].

Therefore, the results indicate higher methyl groups, which could be attributed to the higher content of Ile in camel β-casein primary sequence; the amount of Ile in camel β-casein

(5.55%) is significantly higher when compared with bovine β-casein (4.12%) [START_REF] Salmen | Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia[END_REF]. These results lead to note that camel β-casein is suggested to be more hydrophobic than bovine β-casein. These findings are in agreement with the highest FC values observed with camel milk and with the findings of [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF] and [START_REF] Fernández | Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin[END_REF].

Milk protein denaturation

Denaturation rate

Denaturation Rate (DR, %) values of camel and bovine milk proteins under different heating temperatures (70, 80, 90, and 100 °C for 30min) are shown in Fig. 4a. After heating, the free -SH group concentration of bovine and camel milk rose significantly (P < 0.05) as a function of temperature, with higher contents for camel milk. DR values reached their maximum at 90 °C with values of 75.2 ± 3% and 174.4 ± 15% for bovine and camel milk, respectively. At 100 °C, DR values started to decline to 22.5 ± 5% and 136.9 ±5% for bovine and camel milk, respectively.

These results could be explained by the whey protein denaturation that occurred during the first 30 min of heating at a temperature of 90 °C, regardless of the milk origin.

Furthermore, camel whey proteins are characterised by a higher thermal sensitivity than bovine proteins as reported by Felfoul et al. (2015). β-Lg plays the main role in the aggregation phenomenon of the bovine whey proteins after heating. Indeed, after heating at 70 °C, the free -SH groups of the β-Lg monomers were exposed, leading to the reactivity of this protein toward thiol/disulphide interchange reactions during the heat-treatment. Then, the β-Lg reacts with the α-La, which contains four buried disulphide bridges, forming reactive β-Lg-α-La dimers that react with the other whey molecules leading to the creation of the heat induced aggregates at higher temperatures (> 90 °C) (De la [START_REF] De La Fuente | Recent advances in the characterisation of heat-induced aggregates and intermediates of whey proteins[END_REF].

For camel milk, [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF] noted that the camel whey proteins are characterised by a higher thiol group's concentration after heating at 90 °C for 30min. This behaviour was explained by the denaturing temperature of camel α-La (~73.8 °C) and the presence of camel serum albumin (CSA) whose molecular structure is characterised by the presence of 7 disulphide bridges [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF].

HPLC analysis

The protein composition of camel and bovine milk under native conditions (20 °C)

and after different heat-treatments (70, 80, 90, and 100 °C for 30 min) is presented in Fig. 5.

HPLC chromatograms of unheated bovine and camel milk protein fractions (Fig. 5a and5b, respectively, 20 °C) showed that for cow milk, six major peaks (RT: 19.22 min,23.92 min,25.74 min,27.47 min,27.76 min and 30.37 min) were identified as κ-casein (~7.1%), αcasein (~24.7%), β-casein (~37.4%), α-La (~4.7%), protein fraction F (~1.2%) and β-Lg (~24.9%). Protein fraction (F) is suggested to be dimers of β-Lg [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF].

For the camel milk chromatograms (Fig. 5b, 20 °C), five major protein peaks with retention time (RT) of 19.50 min, 21.63 min, 22,68 min, 25.27 min and 26.93 min were identified. The identification of camel proteins was determined using the chromatograms of camel caseins and whey as it cannot be realised using bovine proteins standards. The proteins with RT of 19.50 and 26.93 min were identified as α-casein (~29.1% of total milk proteins) and β-casein (~44% of total milk proteins), respectively, as the β-casein is the main camel protein in camel milk and caseins fractions (Fig. 5c) and in agreement with previous work [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Kappeler | Sequence analysis of Camelus dromedarius milk caseins[END_REF]. On the other hand, the peak with RT of 21.63 min is the α-La representing 18.8% of total camel milk proteins. Indeed, the α-La is the main whey protein (Fig. 5c) in camel milk as β-Lg is totally absent [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF][START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF]. Finally, both peaks with RT of 22.68 min, 25.27 min were specific protein fractions of camel milk whey fractions representing respectively 3.1% (F1) and 4.7% (F2) of the total amount of camel milk proteins. These protein fractions are suggested to be identified as the peptidoglycan recognition protein (PGRP) and CSA for F1 and F2 respectively in agreement with [START_REF] El-Hatmi | Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum[END_REF], [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF][START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF].

As expected, HPLC chromatograms showed that camel milk proteins exhibited a higher amount of β-casein which can be considered as the main protein of camel milk (representing 44% of the total camel proteins). The results are consistent with the data previously reported by other works [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Hailu | Functional and technological properties of camel milk proteins: a review[END_REF][START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF]. No peaks were detected for the κ-casein, probably due to its low concentration that makes it obscured by other caseins, in agreement with the results of [START_REF] Farah | Vitamin content of camel milk[END_REF].

Furthermore, chromatograms showed that the α-La was the major whey protein in camel milk and represents ~18.8% of the total milk proteins, which was significantly higher than the content of the α-La in bovine milk (~4.7%) in agreement with [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF] who reported that the α-La had the greatest content in soluble camel milk fraction ranging between 5.2 and 19.36%.

To reveal the denaturation and aggregation phenomena of the proteins in both camel and bovine milk, HPLC results (Fig. 5a,b) showed that caseins (peaks corresponding to κcasein, α-casein and β-casein) remained almost intact after heating both camel and bovine milk [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF]. However, the main camel and bovine whey proteins were significantly affected upon heating as function of the themal-treatment temperature. Indeed, the peaks of the α-La and the β-Lg started immediately diminished after the heat-treatment of bovine milk at 80 °C for 30 min. The β-Lg peak totally disappeared after heating at 90 °C and 100 °C during 30 min, unlike the β-Lg dimer peak that increased due to the creation of heatinduced disulphide-bonded dimers as intermediates in the β-Lg aggregation [START_REF] Manderson | Effect of heat treatment on the conformation and aggregation of β-lactoglobulin A, B, and C[END_REF].

For heat-treated bovine milk (Fig. 5a), a new protein fraction peak appeared(F2, RT = 26.65 min). This peak could be attributed to β-Lg multimers. Indeed, after heating at 80 °C, the β-Lg in its monomeric form can associate with other proteins and aggregate. Thus, the quantities of β-Lg monomers decreased and the amount of aggregates larger than trimers increased [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Moro | Effects of heat-treated β-lactoglobulin and its aggregates on foaming properties[END_REF].

The heating temperatures chosen (70, 80, 90 and 100 °C for 30 min) were based on the work of Felfoul et al. (2015b) and [START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF]. These parameters correspond to different stages of denaturation of milk proteins. Indeed, it has been shown by previous studies that at 70 °C, the β-Lg molecules are reduced from dimers to monomers and begin to unfold. Furthermore, the denaturation temperature of the β-Lg in sweet bovine whey is around 80 °C (79.6±0.7 °C) as reported by Felfoul et al. (2015b). On the other hand, the denaturation temperature values of both bovine and camel α-La are near 70 °C. Felfoul et al. (2015b) reported also that 90 °C is the temperature of the total denaturation and aggregation of whey proteins. Finally, we have chosen the heating temperature of 100 °C to make sure that all proteins are already denatured. Heating up time is 30 min was chosen according to previous work, especially that of [START_REF] Lam | The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate[END_REF].

Fig. 5b indicates that thermal treatment of camel milk at 70, 80, 90 and 100 °C for 30 min had no any significant effect on the camel casein fraction and on the whey proteins as CSA and PGRP except for the camel α-La. Indeed, Fig. 5b shows that the peak of the α-La began to decline after the heat-treatment at 70 °C during 30min. Afterwards, it decreased more with further increase of the heating temperature from 80 °C to 100 °C for 30min. The reduction of the chromatograms peaks could be the consequence of the proteins denaturation and/or aggregation under heating. [START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF] confirmed that camel α-La completely disappeared in camel milk heat-treated at 80 °C for 60 min. [START_REF] Atri | Comparative study on heat stability of camel and bovine apo and holo αlactalbumin[END_REF] showed greater thermal stability of the camel α-La than the bovine α-La in its both states: holo-α-La (with calcium) and apo-α-La (without calcium) due to the lack of the β-Lg in camel milk [START_REF] Elagamy | Effect of heat treatment on camel milk proteins with respect to antimicrobial factors: a comparison with cows' and buffalo milk proteins[END_REF].

Electrophoresis patterns of camel and bovine milk

For the native bovine (Fig. 6a, lane 20 °C), seven major proteins bands of 80 kDa, 66 kDa, 35kDa, 30 kDa, 28 kDa, 18 kDa and 14 kDa were identified, corresponding to lactoferrin, BSA (bovine serum albumin), αS-casein, β-casein κ-casein, β-Lg and α-La, respectively. On the other hand, Fig. 6b (lane 20 °C) shows that six major protein bands (80 kDa, 66 kDa, 35kDa, 30kDa, 25kDa and 14 kDa) were identified in camel milk as lactoferrin, CSA, β-casein, αS-casein, κ -casein and α-La. As expected, the most abundant whey protein in bovine milk is β-Lg, whereas this protein is not detected in camel milk. These results are in agreement with previously reported findings [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF][START_REF] Felfoul | Proteomic profiling of camel and cow milk proteins under heat treatment[END_REF][START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF] and with HPLC results (section 3.3.2). Furthermore, camel milk also contains three protein fractions (F) of 60 kDa (F1), 22 kDa (F2) and 19 kDa (F3). These fractions are suggested to be specific components of camel milk whey as reported by [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF].

They are not comparable with any protein in bovine milk. Therefore, F3 is suggested to be identified as the PGRP (19.1 kDa) [START_REF] Kappeler | Expression of the peptidoglycan recognition protein, PGRP, in the lactating mammary gland[END_REF].

Pasteurisation temperature (70 °C) caused no visible modification in both camel and bovine protein gel patterns (Fig. 6, Lane 70 °C) in agreement with the results of Felfoul et al. (2015b). Some faint bands in the region between BSA and caseins appeared to increase in intensity after heating at 80 °C, 90 °C and 100 °C. According to [START_REF] Havea | Heat-induced aggregation of whey proteins: comparison of cheese WPC with acid WPC and relevance of mineral composition[END_REF], these bands were probably intermediate protein species (β-lactoglobulin dimers, trimers) that were formed during milk heating.

Camel α-La band remained constant after heating milk at both temperature values 70 and 80 °C, whereas at 90 °C, α-La, F1 and κ-casein bands decreased, in agreement with the findings of Felfoul et al. (2015b). For bovine milk, the β-Lg and α-La bands remained constant at 70 °C and 80 °C, but started to disappear after the heat treatment temperature of 90 °C in agreement with Felfoul et al. (2015b).

3.4.

Surface characteristics of milk proteins

Surface hydrophobicity

The impact of various thermal treatments (70, 80, 90 and 100 °C for 30 min) on skimmed bovine and camel milk was studied through the changes in the overall surface hydrophobicity of camel and bovine milk protein solutions at the same protein concentration (1g L -1 ; Fig. 4b). Under native conditions (20 °C), the surface hydrophobicity of camel milk protein solution was significantly higher than that of its bovine counterpart (P < 0.05); the bound-BPB amounts at 20 °C were 1.49 ± 0.51 µg mL -1 and 3.40 ± 0.72 µg mL -1 for bovine and camel milk, respectively. These results are consistent with those of [START_REF] Kappeler | Sequence analysis of Camelus dromedarius milk caseins[END_REF]. The differences in the protein proportions in both milk samples and the presence of highly hydrophobic proteins as camel β-casein and α-La in camel milk could explain the higher surface hydrophobicity of the camel milk proteins relative to those in bovine milk [START_REF] Atri | Comparative study on heat stability of camel and bovine apo and holo αlactalbumin[END_REF][START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF].

The surface hydrophobicity of bovine milk proteins increased when the temperature of the heat-treatment rose from 20 °C to 80 °C (bound-BPB = 6.32±0.40 µg mL -1 ), and then it decreased with further increase of temperature from 90 °C to 100 °C (Fig. 4b). After heating bovine milk at 90 °C and 100 °C for 30min, bound-BPB values reached 1.91 ± 0.70 and 0.71± 0.61 µg mL -1 , respectively. As was the case for bovine milk, the surface hydrophobicity of camel milk greatly increased after heating at 70 °C for 30 min and achieved a maximum value of bound-BPB values of 11.16 ± 0.34 µg mL -1 at 80 °C. Finally, it significantly decreased to 5.95 ± 0.62 µg mL -1 at 90 °C and 2.48 ± 0.29 µg mL -1 at 100 °C.

In support of these results, [START_REF] Borcherding | Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk[END_REF] found that higher temperature during heating leads to an increase of the hydrophobic interactions due to an exposure of hydrophobic groups, which are buried inside the globular structure of whey proteins.

Moreover, [START_REF] Lam | The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate[END_REF] noted that the hydrophobic moieties of whey proteins would be buried within the molecular structure of the created aggregates leading to the reduction of the overall hydrophobicity rate of milk proteins. These findings are in agreement with the lowest surface hydrophobicity observed with bovine milk at 90 °C and 100 °C and with the results of HPLC (section 3.2.2).

The highest surface hydrophobicity of camel milk proteins after heating can be explained by the lack of the β-Lg in this milk and also the exceptional hydrophobicity of camel α-La in agreement with the highest FC values of camel milk (section 3.1) and with the results of [START_REF] Atri | Comparative study on heat stability of camel and bovine apo and holo αlactalbumin[END_REF], who found that the purified camel α-La was characterised by the higher surface hydrophobicity that given by its bovine counterpart. Indeed, the primary structure of camel α-La shows greater hydrophobicity of the α-La amino-acid sequence 25-35 in the hydrophobic core.

Determination of ζ-potential

Surface charge (or ζ-potential) of bovine and camel milk (at a protein concentration of 1 g L -1 ) as a function of temperature pre-treatments (20, 70, 80, 90, and 100 °C for 30 min)

were measured and are given in Fig. 7a. Overall, the ζ-potential values of camel milk were significantly lower than that of their bovine counterparts (P <0.05) regardless of heating temperature value except after heating at 90 °C and 100 °C for 30min.

Under native conditions, the ζ-potential values were -22.6 ± 0.8 mV and -19.9 ± 0.6 mV for bovine and camel milk, respectively. This is consistent with the results of [START_REF] Momen | Effect of dry heating on physico-chemical, functional properties and digestibility of camel whey protein[END_REF] and [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF]. These authors found that the ζ-potential values of camel whey were significantly lower than those of bovine whey proteins isolate solutions and suggested that this difference can be associated with the difference the protein composition of both wheys, the presence of highly basic protein in camel whey, lactoferrin (isoelectric point, pI, = 8.8), and the difference in the pI of 5.01 and 4.2 for camel and bovine α-La (5.01 and 4.2, respectively).

Fig. 7a also shows that camel and bovine milk appeared less negatively charged after a heat-treatment at 70, 80, 90 and 100 °C for 30 min. Thermal treatment at 70 °C for 30min induced a significant decrease of the electronegative charge from -22.6 ± 0.8 mV to -19.6 ± 0.8 mV and from -19.9 ± 0.6 to -18 ± 0.5 mV bovine and camel milk respectively (P < 0.05).

No significant difference was observed between camel and bovine milk after heating at 90 °C and 100 °C for 30min (ζ-potential -16.5 mV). This behaviour can be explained by the denaturation and aggregation of the different milk proteins that may play an important role in this change of electronegative charge.

The same trends were reported for bovine milk by [START_REF] Borcherding | Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk[END_REF] who observed that the heat-treatment of milk induces a decrease in its pH value leading to a lower protein negative charge and therefore a lower electrostatic repulsive forces as a result of heating. On the other hand, [START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF] reported that the acid camel whey carried less negative charge after heating at 90 °C for 30 min due to the dominance of the α-La in camel whey and its aggregation after a thermal treatment at this temperature.

Determination of the interfacial tension of milk

The average interfacial tension values (γ) of camel and bovine milk at a protein concentration of 1 g L -1 after thermal treatment in the temperature range 70-100 °C for 30 min are shown in Fig. 7b. Camel and bovine milk proteins significantly reduced the interfacial tension of the air-water interface from 72 mN m -1 (P < 0.05), which was estimated to be close to the surface tension value of pure water. The surface tension values for camel and bovine milk were in good agreement with those obtained by [START_REF] Kamath | The influence of temperature on the foaming of milk[END_REF] and by [START_REF] Bertsch | Surface tension of whole and skim-milk between 18 and 135C[END_REF] for raw and pasteurised bovine milk.

The surface tension values of camel milk were significantly lower than that of bovine milk (P < 0.05) regardless of heating temperature value (Fig. 7b). Thus, in native conditions (20 °C), the order of effectiveness was camel milk (γ = 43.4 ± 1.4 mN m -1 ) > bovine milk (γ = 32.8 ± 1.7mN m -1 ).

Fig. 7b shows also that the heat-treatments in the temperature range 70 °C to 100 °C for 30 min was found to improve the ability to reduce the interfacial tension at the air-water interface for both camel and bovine milk. Furthermore, a greater efficiency to reduce the surface tension values was attributed to the milk treated at higher temperature values. Indeed, a thermal treatment at 70 °C for 30 min induced a significant decrease of the surface tension from 43.4 to 36.8 mN m -1 and from 32.8 to 28.9 mN m -1 for bovine and camel milk, respectively (P < 0.05).

After a heat-treatment of 90 °C for 30 min, both milk samples retained the best interfacial properties when compared with the other thermal treatments. Hence, the order of effectiveness at this temperature value was camel milk (γ = 21.2 ± 0.6 mN m -1 ) > bovine milk (γ = 31.2 ± 1.2 mN m -1 ). No significant change was found on the evolution of the surface tension values of camel and bovine milk between 90 °C and 100 °C. This behaviour was explained by the increase of the number of hydrophobic interactions after heating due to an exposure of proteins hydrophobic groups, which are buried inside the globular whey proteins at temperature values below 40 °C [START_REF] Borcherding | Effect of foaming temperature and varying time/temperature-conditions of pre-heating on the foaming properties of skimmed milk[END_REF].

The behaviour of native camel and bovine milk can be explained by the higher content of β-casein, which is in agreement with previous foaming properties (section 3.1).

Purified camel β-casein is more efficient in reducing the surface tension at the air-water interface than its bovine counterpart because of the difference in the amino-acid residue composition [START_REF] Lajnaf | Foaming and adsorption behavior of bovine and camel proteins mixed layers at the air/water interface[END_REF]. Heat-treatment of the isolated camel α-La at 90 °C for 30 min and at pH 6.5 also significantly improved the ability of this protein to reduce the interfacial tension at the air-water interface, contrary to acidic pH values (pH 4.3), where the α-La was found to aggregate under heating leading to the reduction of its tensioactive properties [START_REF] Lajnaf | The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk[END_REF].

Conclusion

This study investigated the foaming and physicochemical properties of camel milk proteins as a function of heating temperature (70, 80, 90 and 100 °C). Our findings confirmed the exceptional foaming properties of camel milk and highlighted the importance of the protein composition and their denaturation and/or aggregation state. These observations were related to the high β-casein amount in camel milk as well as its different secondary structure, especially in the β-sheet conformation and its high hydrophobicity as confirmed by FT-IR and 1 H-NMR results. On the other hand, the camel milk proteins were found to have the lowest values of foam stability because of the lowest amount of the κ-casein and the absence of β-Lg in camel milk. In addition, the results of this study indicated that heat-treatment improved the foaming capacity and stability of camel milk proteins. Indeed, the heattreatment affected the physicochemical properties of camel milk proteins with an increase of surface hydrophobicity and a slight decrease of their negative ζ-potential and the interfacial tension values. These findings can be justified by the denaturation and/or aggregation of camel milk proteins after heating in consistence with the DR, RP-HPLC and SDS-PAGE results.

  Figure 5

Acknowledgements

This work was supported by the Alimentary Analysis Unit. We are greatly indebted to Professor A. Ben Ali for editing the manuscript.


Plamen Bokov

Boris Matrot

Noria Medjahdi

Souham Boureghda

Mohamed Essalhi

Eric Konofal

Michel Lecendreux

Jorge Gallego

Christophe Delclaux
email: christophe.delclaux@aphp.fr

Christophe Pr

Delclaux

Cross-sectional study of loop gain abnormalities in childhood obstructive sleep apnea syndrome

Keywords: sleep apnea, childhood, ventilatory control, loop gain Highlights

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

Introduction

Obstructive sleep apnea (OSA), a prevalent disease in childhood [START_REF] Kaditis | Obstructive sleep disordered breathing in 2-to 18-year-old children: diagnosis and management[END_REF], is mediated by a combination of anatomic and neuromotor factors, including ventilatory control [START_REF] Marcus | The obstructive sleep apnoea syndrome in adolescents[END_REF]. Loop gain (LG) is a dimensionless value quantifying the stability of the ventilatory control system; it represents the sensitivity of the feedback loop controlling ventilation and is defined as the size of a corrective ventilatory response relative to the size of the ventilatory disturbance that elicits the correction. The corrective response to a ventilatory disturbance is determined not only by chemoreceptor sensitivity or controller gain (CG) but also by plant gain (PG), which quantifies the extent to which changes in ventilation affect changes in pulmonary capillary blood gas tensions. LG and its components, CG and PG, are frequency-dependent parameters that can be accurately estimated from data-driven dynamic mathematical models [START_REF] Bokov | Comparison of different methods of chemical loop gain measurement during tidal ventilation in awake healthy subjects[END_REF]. Unstable ventilatory control is characterized by high LG and may contribute to cyclical airway obstruction by promoting airway collapse during periods of low ventilatory drive.

It has been stated by Deacon-Diaz and Malhotra in a recent review that "although much evidence supports that unstable chemoreflex control contributes to propagating apneas in OSA, whether loop gain is elevated in OSA, and whether high controller or plant gain contribute to high loop gain, is currently unclear" (4). This issue is important because therapeutic approaches to OSA aimed at lowering both plant and loop gains are currently being investigated [START_REF] Eckert | Phenotypic approaches to obstructive sleep apnoea -New pathways for targeted therapy[END_REF]. Thus, the respective contributions of CG and PG to LG deserve to be studied in children with OSA.

In otherwise healthy children, Armoni Domany and colleagues recently showed that children with OSA had higher PG and lower CG at baseline compared to healthy controls, resulting in similar LG [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF]. It has also been stated that LG could be elevated in untreated OSA patients due to the combined effects of intermittent hypoxia-induced ventilatory long-term facilitation and increased hypoxic sensitivity (4); thus, the LG for oxygen also deserves investigation.

The aim of this study was to assess whether an increased PG could increase LG in a sample of consecutive otherwise healthy children referred for suspicion of OSA. Gains (loop, controller, and plant) were calculated using methods that involved spontaneous variations in ventilation and expiratory gases during wakefulness [START_REF] Bokov | Comparison of different methods of chemical loop gain measurement during tidal ventilation in awake healthy subjects[END_REF]. Three analytical models, two autoregressive, unconstrained models (one bivariate for LG and one trivariate for O2 and CO2 LG) and one constrained model for LG in order to assess the consistency of the analytical methods, were used to better explain the underlying components of the modifications of LG and its components that were expected and to assess whether increased hypoxic sensitivity could be observed. The main objective of our study was to assess whether an increase in LG and its components (PG and CG) can be evidenced in moderate to severe OSA children as compared to children without OSA or with mild OSA since previous studies showing an increased LG involved mainly adults with moderate to severe OSA [START_REF] Deacon-Diaz | Daytime loop gain is elevated in obstructive sleep apnea but not reduced by CPAP treatment[END_REF][START_REF] Gederi | Model-based estimation of loop gain using spontaneous breathing: a validation study[END_REF][START_REF] Hudgel | Instability of ventilatory control in patients with obstructive sleep apnea[END_REF].

Material and Methods

Patients

The inclusion criteria were symptoms of upper airway obstruction (snoring, apnea, restless sleep, oral breathing) in otherwise healthy children with or without obesity.

Non-inclusion criteria were midface deficiency, marked mandibular hypoplasia, prematurity (based on a report of birth at least 4 weeks early), genetic disorders and ongoing treatment for OSA.

Exclusion criteria were the inability to perform tidal breathing recording, an insufficient number of breaths for analysis and failure of computation for all models.

This study was approved by our local Ethics Committee (PHENOSAS: N° 2018-416), and the database of collected data was declared to the French regulatory agency (CNIL). The subjects and their parents were informed of the collection of their prospective data for research purposes, and they could request to be exempted from this study in accordance with French law (non-interventional observational research). This study was non-interventional since the recording of the CO2 response is part of our usual checkup when suspecting OSA (act of health insurance: GLQP006).

Tidal breathing measurements

All recordings started between 7:00 and 8:00 p.m., before polysomnography, in order to avoid the nycthemeral cycling of LG. Recordings of tidal breathing were performed as previously described (3), lasting 20 min, with the first 5 min being discarded. During the recordings, subjects were awake and lying in their beds in a calm and non-stimulating atmosphere. Flow rate, end-tidal PO2 (PETO2) and end-tidal PCO2 (PETCO2) were continuously monitored, and signals were digitized using the MP-100 system (Biopac System Inc., Santa Barbara, CA) at a rate of 50 Hz; these were stored for further analysis.

Loop gain models

The models were fitted on the changes from baseline (mean) levels of the ventilatory parameters ( E V When not specified, the gains given are those for CO2 (the gains for oxygen are always specified). Since tidal breathing measurements were done while awake, the analyses were specifically related to chemosensitivity. It is known that changes in pharyngeal patency that occur in parallel with PCO2 will raise CG. In this case, the overall controller gain (chemoresponsiveness) is equal to the intrinsic gain (chemosensitivity) multiplied by the effectiveness of the upper airways that were not assessed.

In-laboratory polysomnography

Polysomnography studies were performed overnight. An Alice 6 LDx polysomnography system (Philips, Murrysville, PA) recorded the following parameters: chest and abdominal wall motion using respiratory inductance plethysmography, heart rate by electrocardiogram, arterial oxygen saturation by pulse oximetry, transcutaneous PaCO2, airflow using a 3pronged thermistor, nasal pressure by a pressure transducer, electroencephalographic leads (C3/A2, C4/A1, F3A2, F4A1, O1/A2, O2/A1), left and right electrooculograms, submental electromyogram, and tibial electromyogram. Study participants were also recorded with an infrared video camera. Experienced pediatric sleep physicians scored patients using standard pediatric sleep scoring criteria, as previously done [START_REF] Bokov | The utility of acoustic pharyngometry and rhinometry in pediatric obstructive sleep apnea syndrome[END_REF]. The definition of OSA was sleep disorder breathing symptoms and an obstructive apnea hypopnea index (OAHI) ≥2 episode.h -1

(1). Based on the OAHI, patients were further classified as having mild (2 ≤ OAHI < 5) or moderate to severe (OAHI ≥ 5) OSA [START_REF] Bokov | The utility of acoustic pharyngometry and rhinometry in pediatric obstructive sleep apnea syndrome[END_REF].

Statistics

Sample size calculation. In our study of healthy adult subjects, the normal value of unconstrained bivariate model LG was 0.136 ± 0.107 [START_REF] Bokov | Comparison of different methods of chemical loop gain measurement during tidal ventilation in awake healthy subjects[END_REF]. The objective of our study was to assess whether children with OSA were characterized by an increased LG. Setting this increase at 0.085 (by ~60%), with a type I error rate of 0.05 (two-tailed) and a type II error rate of 0.20, we had to include 26 children in each group (OAHI <5.h -1 and ≥5.h -1 : calculated using the T statistic and non-centrality parameter). Thus, at least 54 children had to be included in the study.

Results were expressed as medians [25 th -75 th percentiles] due to the non-normal distribution of gain values. Comparisons of continuous variables between the groups were performed using the Mann-Whitney U test. The different groups evaluated were children with OAHI <5.h -1 versus those with ≥5.h -1 (main objective), and children with OSA (OAHI≥2.h -1 ) versus those without OSA (OAHI<2.h -1 ) (descriptive objective). Categorical variables were compared using the chi-square test. Correlations were evaluated using Pearson's correlation coefficient. The other statistical tests are described in the text. A P-value <0.05 was deemed significant. No correction for multiple testing was done due to the pathophysiological design of the study [START_REF] Rothman | No adjustments are needed for multiple comparisons[END_REF]. All statistical analyses were performed with Statview 5.0 software (SAS institute, Cary, North Carolina, USA).

Results

Failure p=0.047), while the time constants were not different. Circulatory delays were not different (see Figure 3 legend).

Finally, the unconstrained trivariate model was utilized to assess the respective contributions of both CO2 and O2 chemosensitivities. Figure 4 shows the results of oxygen LG (medium frequency) and its components (CG and PG for O2) in children without OSA or mild OSA compared to those with moderate to severe OSA. The figure shows that the gains for oxygen

were not increased in children with moderate to severe OSA. Similar results for CO2 gains were observed with the trivariate unconstrained model compared with the two bivariate models (data not shown).

Total LG was determined by both O2 and CO2 LGs and logically correlated to each gain. In a multivariate analysis with total LG (medium frequencies) as a dependent variable and O2 and CO2

LGs as independent variables, only O2 LG remained independently correlated with total

LG (p=0.006, r 2 =0.47) in the children with OSA (n=19). In the non-OSA group (n=35), only

CO2
LG was independently linked to total LG (p<0.001, r 2 =0.56).

Discussion

In this study, we showed that children with moderate to severe OSA had not an increased LG as compared to those without OSA or with mild OSA, children with OSA had a higher PG compared to children without OSA, and PG was positively associated with the severity of OSA. The higher PG in children with OSA was related to an increase in steady-state PG, while no modification of CG was observed. Additionally, we demonstrated no argument for increased hypoxic sensitivity.

Although OSA is often related to adenotonsillar hypertrophy in children, this effect is generally associated with underlying abnormalities of upper airway function [START_REF] Marcus | The obstructive sleep apnoea syndrome in adolescents[END_REF]. OSA involves functional factors related to control of breathing (CG) and the effectiveness of gas exchange (PG), which is suggestive of abnormal LG. However, whether LG and its components are consistently modified in OSA patients remains debated (4). We previously reported that OSA was associated with increased PG, but not increased LG, in obese women [START_REF] Bokov | Loop gain in severely obese women with obstructive sleep apnoea[END_REF]. The present study extends these results to otherwise healthy younger patients. Moreover, we found a positive relationship between PG and AHI, which is in line with previous studies (with a similar degree of explained variance) [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF][START_REF] Domany | Cardioventilatory Control in Preterm-born Children and the Risk of Obstructive Sleep Apnea[END_REF]. We also found that AHI was independently linked to the BMI z-score and PG, and the time constant of PG, which is linked to lung CO2 stores, was similar in children with and without OSA. Thus, the effect of obesity on lung stores as a cause of increased PG can be definitively ruled out, as previously suggested [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF][START_REF] Bokov | Loop gain in severely obese women with obstructive sleep apnoea[END_REF]. The bivariate constrained model further demonstrated that the increase in PG was due to an increase in steady-state PG, which is a novel result. This steady-state PG is proportional to alveolar PCO2 and inversely proportional to alveolar ventilation and cardiac output ( 14). Accordingly, it may be anticipated that children suffering from OSA would show a mild degree of alveolar hypoventilation, as evidenced in adult OSA [START_REF] Zerah-Lancner | Pulmonary function in obese snorers with or without sleep apnea syndrome[END_REF]. Interestingly, in our children with OSA, PETCO2 was not increased during wakefulness (Table 1), while more pronounced nocturnal hypoventilation was evidenced in moderate to severe OSA. This increase may be related to modifications of blood/tissue CO2 stores [START_REF] Khoo | Factors inducing periodic breathing in humans: a general model[END_REF], which warrants further investigation.

Armoni Domany and colleagues showed that PG was normalized following adenotonsillectomy, suggesting that high PG values were a consequence of OSA [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF].

Furthermore, the reported resolution of complex sleep apnea and the amelioration of ventilatory control abnormalities after positive pressure therapy suggested that OSA may lead to ventilatory control abnormalities [START_REF] Orr | Pathogenesis of central and complex sleep apnoea[END_REF]. Thus, chemical control instability may not only be a consequence but also a cause of obstructive apnea as detailed below.

Medium frequency LG values > 1 are associated with major respiratory instability but have not been observed in OSA patients to the best of our knowledge. Based on this assumption, it is interesting to point out that loop gain and its component, plant gain, can favor an obstructive event following an apnea by at least two mechanisms.

First, after an apnea, the LG will produce a ventilatory overshoot of magnitude LG*Veupnea, with Veupnea being the eupneic ventilation. This overshoot will itself induce a state with reduced ventilation [Veupnea*(1-LG 2 )]. If the reduction of the ventilation exceeds 30% of Veupnea, this event will be scored as a hypopnea. Hence, if loop gain exceeds a threshold value of 0.3 1/2 (≈0.55), a hypopnea can potentially be induced by an apnea, and a substantial reduction of the LG would theoretically at least divide the AHI by a factor of 2. Among our subjects with OSA, no child presented with a value of medium frequency LG higher than 0.55 either with the unconstrained or constrained bivariate models. Thus, in our opinion, the notion that a relative increase in LG (less stable LG) in OSA contributes to apnea-hypopnea genesis remains speculative.

The second mechanism by which an apnea could induce an obstructive event is by reduced ventilatory drive to the upper airways leading to obstructive and/or central events. After an apnea, the ventilatory overshoot is still given by LG*Veupnea. This modification of the ventilation will decrease alveolar CO2 (relative hypocapnia) that scales as PG*LG*Veupnea.

This relative hypocapnia will induce a decrease in the drive of the upper airway muscles and will consequently increase the upper airway resistance, leading to an obstructive event. Sleep also unmasks a highly sensitive hypocapnia-induced apneic threshold, whereby apnea is initiated by small transient reductions in PaCO2 below eupnea and respiratory rhythm is not restored until PaCO2 has risen significantly above the eupneic level. It has been proposed that the CO2 reserve (i.e., the difference in PaCO2 between eupnea and the apneic threshold) when combined with PG and CG are the key determinants of breathing instability in sleep. The CO2 reserve varies inversely with PG, regardless of the stimulus and whether it acts primarily at the peripheral or central chemoreceptors [START_REF] Dempsey | The ventilatory responsiveness to CO(2) below eupnoea as a determinant of ventilatory stability in sleep[END_REF]. Thus, an increased PG will further decrease PaCO2 to the extent that ventilatory drive is decreased and favors both central and obstructive apneas. As a consequence, one may also hypothesize that a high PG per se (independently of

LG) will contribute to the pathophysiology of apnea. In our opinion, the contribution of the second mechanism to airway instability in OSA is probably the most important mechanism, justifying the causal relationship between PG and AHI and the usual beneficial effects of therapies directed at reducing PG.

Finally, the results of LG and its components in the group without OSA can be regarded as normal values. Overall, it seems that both PG and LG are reduced in these children as compared to healthy adults (3). More stable ventilatory control may contribute to the lower normal AHI value (<1.h -1 ) of children as compared to adults (<5.h -1 ).

The unconstrained trivariate model allowed the exploration of O2 chemosensitivity. CO2 is considered the primary stimulus to chemoreflex control; accordingly, in children without OSA, total LG was independently linked to the sole CO2 LG. In contrast, in children with OSA, oxygen LG was the main contributor to total gain, suggesting a pathophysiological regulation. It has been shown that if the additional stimuli of hypoxia is combined with hypercapnia, such as during apneas, the contribution of the peripheral chemoreceptor drive to ventilation will increase and the gain of the ventilatory response to CO2 will increase further; a relationship described as hypoxic sensitization of the peripheral chemoreflex response to CO2 [START_REF] Duffin | Measuring the ventilatory response to hypoxia[END_REF]. Nevertheless, we did not observe any result arguing for such a phenomenon since controller gains were similar whatever the group. This result seems at odds with the recent findings of Alex and colleagues who have demonstrated that exposure to mild intermittent hypoxia increases LG in OSA and have suggested that it could be due to enhanced CG [START_REF] Alex | Exposure to mild intermittent hypoxia increases loop gain and the arousal threshold in participants with obstructive sleep apnoea[END_REF].

The increase in PG in children with OSA may have therapeutic implications. Respiratory stimulants (e.g., inhaled CO2, rebreathing, acetazolamide, and theophylline) act to increase CO2 damping and reduce PG (due to the reduction in the difference between alveolar and inspired PCO2). Accordingly, did not show any difference in loop gain values (mean ± SD = 0.41 ± 0.09 and 0.41 ± 0.12, respectively; no trend is evidenced) [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF]. Nevertheless, an increase in LG in a specific endotype, as evidenced in adults [START_REF] Eckert | Defining phenotypic causes of obstructive sleep apnea. Identification of novel therapeutic targets[END_REF], cannot be ruled out. Second, our tidal ventilation recordings were made during wakefulness, while an impairment of ventilatory CO2 response, suggesting impaired chemosensitivity and controller gain, seems specific to a sleep state (2), even though Armoni Domany and colleagues observed similar results to ours during sleep [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF].

Our study also has strengths. First, we used an additional bivariate constrained model to assess the consistency between models. As previously demonstrated in healthy subjects (3), the two bivariate models gave similar results; this finding is important because part of the discrepancies of the literature could have been due to different modeling approaches. We confirmed the results of Armoni Domany and colleagues using both 1) a bivariate unconstrained model (standard autoregressive model) (while they used a non-standard autoregressive model) [START_REF] Domany | The Effect of Adenotonsillectomy on Ventilatory Control in Children with Obstructive Sleep Apnea[END_REF] and 2) a bivariate constrained model. Second, the bivariate constrained model allowed us to ascribe the increase in PG to an increase in steady-state plant gain, which eliminated the role of lung CO2 reserve.

In conclusion, in children with OSA, plant gain was higher compared to healthy controls and positively associated with AHI. Ventilatory control abnormalities, mainly the extent to which changes in ventilation affect changes in pulmonary capillary blood gas tensions, may contribute to OSA pathophysiology. The constrained model allowed computing of the steady-state controller gain (CG) and plant gain (PG) (upper panel) and the time constant for both controller and plant gains. The children with an OAHI ≥5.h -1 were characterized by a higher steady-state PG (more negative values) as compared to those with OAHI <5.h -1 . These two groups of children had similar circulatory delays, whether expressed as seconds (6.20 [3.76; 8.94

Figure 1 .

 1 Figure 1. Loop gain and its components according to the severity of OSA.

Figure 2 .

 2 Figure 2. Linear correlation between the apnea-hypopnea index (AHI) and plant gain

Figure 3 .

 3 Figure 3. Steady-state gains and time constants determined with the bivariate constrained

Figure 4 .

 4 Figure 4. Loop, controller and plant gains for oxygen according to the severity of OSA.

  to obtain reliable recordings of resting ventilation (inability to perform tidal breathing

	those without OSA or with mild OSA (43.5 mmHg [43.0; 44.7], n=10 versus 42.0 mmHg
	[41.0; 43.2], n=29, respectively; p=0.041).
	Figure 2 shows the correlations between AHI and PG. We chose AHI rather than OAHI, since
	recording [n=4], an insufficient number of breaths for analysis [n=2], inability to support the an increase in gain may favor both obstructive and central apneas. Whatever the model, a
	face mask [n=6], and leaks [n=2]) was observed in 14/68 children (21%). Failure to obtain similar positive correlation was observed between AHI and PG. Further correlation analyzes
	recordings was not related to age. Failure of computation was observed for 4/54 patients in showed that BMI z-score (R=0.26, p=0.039) and LG (medium frequency, bivariate
	the constrained bivariate model; no failures were observed for both the unconstrained constrained model) (R=0.32, p=0.019) were linearly correlated with AHI.
	bivariate and trivariate models. A multivariate analysis with AHI as the dependent variable and BMI z-score, PG and LG
	(medium frequency) as the independent variables demonstrated that BMI z-score (p=0.023)
	and PG (p=0.046) remained independently related to AHI (r 2 of the model=0.25, p=0.003).

Table

1

describes the characteristics of included and excluded children. There was no obvious selection bias, the included and excluded children had similar characteristics. OSA was observed in 35% (19/54) of included children, and moderate to severe OSA was observed in 31% (17/54) of included children.

Table

2

shows the comparisons between children with and without OSA and the results of LG analysis with the unconstrained bivariate model, which had no failure rate. PG was significantly increased in children with OSA compared with children without OSA.

Then, we analyzed whether the moderate to severe children, requiring OSA treatment (1), differed from the less severe children. Figure

1

shows the results of LG (medium frequency) and its components (CG and PG) with the two bivariate models of children without OSA or mild OSA as compared to those with moderate to severe OSA. Overall, the two models showed similar results; PG was increased in children with moderate to severe OSA. Children without OSA or with mild OSA had similar CG compared to children with moderate to severe OSA. Valid data of transcutaneous PCO2 were obtained in 39/54 children (72%). The children with moderate to severe OSA were characterized by a higher nocturnal PaCO2 as compared to

The constrained bivariate model allowed the evaluation of the underlying mechanisms of the increased PG in children with OSA. Figure

3

shows the steady-state and time constant values for both CG and PG. An increased steady-state PG (higher negative value) was demonstrated in children with moderate to severe OSA (-0.97 [-1.16; -0.61] versus -0.58 [-0.97; -0.37],

  Xie and colleagues have shown that hypercapnic rebreathing reduced AHI to 15 ± 4% of control in most OSA subjects (17/21)[START_REF] Xie | Effects of stabilizing or increasing respiratory motor outputs on obstructive sleep apnea[END_REF], further suggesting that increased PG is prevalent in OSA, probably independently of the value of LG.Our study has limitations. First, our study is mainly descriptive. Nevertheless, the results of the different studies of LG in patients suffering from OSA are conflicting, as recently reviewed by Deacon-Diaz and Malhotra (4). Thus, the confirmation of the relationship between PG and AHI deserved to be done and is an important finding. An increase in loop gain has not been found that may be related to underpowered sample sizes since a new sample size calculation based on the present results shows that at least 368 children would have been necessary for such a demonstration. Nevertheless, the recent study of Armoni Domany et al.

	included 99 otherwise healthy children (53 with OSA [OAHI>1/h] and 46 without OSA) and

Table 1 .

 1 Characteristics of the included children.

	Characteristics	Excluded children	Analysed children	P
		N=14	N=54	value#
	Age, years	10.1 [5.7; 11.9]	11.6 [8.1; 15.2]	0.156
	Sex, female/male, n	4/10	19/35	0.758
	Ethnicity: C/B/A/M, n &	13/0/0/1	35/11/3/5	0.177
	Height, m	139 [116; 154]	152 [132; 165]	0.104
	Weight, cm	30.7 [21.5; 65.0]	51.0 [27.0; 61.4]	0.240
	BMI z-score	+0.3 [-0.4; +2.0]	+0.5 [-0.3; +1.7]	0.773
	Polysomnography
	AHI.h -1	1.8 [1.1; 8.7]	1.5 [0.7; 7.3]	0.779
	OAHI.h -1	1.1 [0.7; 7.1]	1.0 [0.3; 5.6]	0.651
	OAHI≥2.h -1 , n	5	19	0.999
	OAHI≥5.h -1 , n	5	17	0.999
	ODI.h -1	1.7 [0.6; 4.1]	1.4 [0.5; 5.4]	0.809
	Resting ventilation
	Cycle duration, s		3.2 [2.7 ; 3.8]
	VT, mL		337 [275 ; 440]
	V'E, L.min -1		6.56 [5.38 ; 7.67]
	VD, mL		115 [80 ; 150]
	PETCO2, mmHg		37.2 [35.0 ; 39.7]
	PETO2, mmHg		101 [98 ; 104]
	ODI is oxygenation desaturation index; AHI is apnea-hypopnea index; OAHI is obstructive
	AHI ; BMI is body mass index ; VT is Tidal volume ; V'E is minute ventilation ; VD is
	volume of dead space : PETCO2 is end-tidal CO2.
	& : C denotes Caucasian, B denotes Sub-Saharan African/Caribbean, A denotes Asian and M
	denotes mixed ethnicity

# : comparison of excluded and included children using Mann Whitney U test or chi two test

Table 2 .

 2 Characteristics of the analysed children according to the presence or absence of OSA.

	Characteristics	Without OSA	With OSA	P value
		(OAHI<2.h -1 ), N=35	(OAHI≥2.h -1 ), N=19
	Age, years	11.9 [7.6; 15.8]	10.6 [9.2; 13.9]	0.612
	Sex, female/male, n	15/20	4/15	0.142
	Ethnicity: C/B/A/M, n &	25/6/2/2	10/5/1/3	0.469
	Height, m	148 [128; 165]	152 [140; 164]	0.568
	Weight, cm	46.9 [25.7; 59.0]	61.0 [36.8; 77.5]	0.054
	BMI z-score	0.4 [-0.4; +0.9]	2.0 [-0.1; +2.3]	0.011
	OAHI≥5.h -1 , n	0	17
	Unconstrained bivariate model
	Plant Gain, mmHg.min.L -1	0.17 [0.06; 0.26]	0.26 [0.15; 0.36]	0.036
	Controller Gain, L.mmHg.min -1	0.37 [0.23; 0.54]	0.36 [0.31; 0.56]	0.772
	Loop Gain, low frequencies	0.057 [0.019; 0.243]	0.121 [0.069; 0.198]	0.221
	Loop Gain, medium frequencies	0.076 [0.019; 0.128]	0.104 [0.073; 0.140]	0.205
	Loop Gain high frequencies	0.040 [0.011; 0.085]	0.044 [0.027; 0.068]	0.737
	OSA is obstructive sleep apnea; BMI is body mass index; AHI is apnea-hypopnea index

& : C denotes Caucasian, B denotes Sub-Saharan African/Caribbean, A denotes Asian and M denotes mixed ethnicity

  ] versus 7.44[3.77; 12.57], respectively; p=0.489) or number of breaths (1.8 [1.0; 2.6] versus 2.0 [1.2; 3.9], respectively; p=0.315).

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.