Anthony Sehl

Leslie Couëdelo

Ikram Chamekh-Coelho

Carole Vaysse

Maud Cansell
email: mcansell@enscbp.fr

In vitro lipolysis and lymphatic absorption of n-3 long-chain polyunsaturated fatty acids in the rat: influence of the molecular lipid species as carrier

Keywords: n-3 polyunsaturated fatty acids, triglycerides, phospholipids, bioavailability, lymph Abbreviations

come

In vitro lipolysis and lymphatic absorption of n-3 long-chain polyunsaturated fatty acids in the rat: influence of the molecular lipid species as carrier

INTRODUCTION

Numerous studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), are associated with a reduced risk of developing several pathologies such as cardiovascular [START_REF] Riediger | A systemic review of the roles of n-3 fatty acids in health and disease[END_REF] , neurodegenerative [START_REF] Dyall | Neurological benefits of omega-3 fatty acids[END_REF] , inflammatory [START_REF] Calder | Omega-3 fatty acids and inflammatory processes: from molecules to man[END_REF] diseases, and cancer [START_REF] Saini | Omega-3 and omega-6 polyunsaturated fatty acids: Dietary sources, metabolism, and significance -A review[END_REF] . Indeed, EPA and DHA are directly implicated in lipid metabolism by lowering triglyceridemia [START_REF] Simopoulos | An increase in the omega-6/omega-3 fatty acid ratio increases the risk for obesity[END_REF] but they are also precursors of eicosanoids with strong anti-inflammatory properties [START_REF] Calder | Omega-3 fatty acids and inflammatory processes: from molecules to man[END_REF][START_REF] Zarate | Significance of long chain polyunsaturated fatty acids in human health[END_REF] . Results from the latest French epidemiological study [START_REF] Dubuisson | The French dietary survey on the general population (INCA3) -French Agency on Food, Environmental and Occupational Health and Safety[END_REF] have shown that, even if the consumption of total lipids was in line with the recommendations (34% of the total energy intake for 35-40% recommended), EPA and DHA consumption was at least 32% lower than the French guidelines (117 and 169 mg per day, respectively, for recommendations of 250 mg per day of each n-3 long-chain PUFA (n-3 LC-PUFA)) [START_REF] Astorg | Actualisation des apports nutritionnels conseillés pour les acides gras -Version tégra t les mod f cat o s apportés par l'erratum du 8 ju llet 9. 7) v s et rapport de l' ses sur[END_REF]9) . In order to cover the n-3 LC-PUFA physiological needs, increasing EPA and DHA intakes represents a major nutritional challenge. One of the strategies consists in improving their bioavailability, without changing the total lipid intake. Several studies have demonstrated that lipid bioavailability depends on physicochemical factors of the dietary intake [START_REF] Cansell | Marine phospholipids as dietary carriers of long-chain polyunsaturated fatty acids[END_REF][START_REF] Couëdelo | Impact of various emulsifiers on ALA bioavailability and chylomicron synthesis through changes in gastrointestinal lipolysis[END_REF][START_REF] Couëdelo | Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion[END_REF][START_REF] Garaiova | A randomised cross-over trial in healthy adults indicating improved absorption of omega-3 fatty acids by pre-emulsification[END_REF][START_REF] Michalski | Multiscale structures of lipids in foods as parameters affecting fatty acid bioavailability and lipid metabolism[END_REF] . Indeed, nowadays, EPA and DHA are essentially consumed as oily fish for food intake or as fish oil (mainly triglycerides (TG) or ethyl esters (EE)) in food supplements. In the case of food supplements, the interest for marine phospholipids (PL) has recently increased. However, the limited data concerning the impact of the molecular lipid species (TG vs PL) of lipid intake on the n-3 LC-PUFA fate, leads to conflicting results. On the one hand, n-3 LC-PUFA levels were increased in the liver and brain of rats fed EPA and DHA in the form of PL compared to TG [START_REF] Graf | Age dependent incorporation of 14C-DHA into rat brain and body tissues after dosing various 14C-DHA-esters[END_REF][START_REF] Rossmeisl | Metabolic effects of n-3 PUFA as phospholipids are superior to triglycerides in mice fed a high-fat diet: possible role of endocannabinoids[END_REF] . On the other hand, accretion of DHA in rat liver, plasma and kidney was significantly lower when it was provided as PL [START_REF] Song | Polyunsaturated (n-3) fatty acids susceptible to peroxidation are increased in plasma and tissue lipids of rats fed docosahexaenoic acid-containing oils[END_REF][START_REF] Song | Enhanced level of n-3 fatty acid in membrane phospholipids induces lipid peroxidation in rats fed dietary docosahexaenoic acid oil[END_REF] . Human studies were even less conclusive. In a double-blind, randomized, placebo-controlled, crossover study, a 4 week-consumption of 600 mg of n-3 LC-PUFA in PL increased the n-3 LC-PUFA levels in plasma and red blood cells (RBC) compared with the TG form [START_REF] Ramprasath | Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil[END_REF] . In contrast, no effect of the molecular species of the dietary lipids (TG vs PL) was observed on n-3 LC-PUFA levels in plasma and RBC after 28 consecutive days of supplementation with 816 mg EPA per day and 522 mg DHA per day [START_REF] Yurko-Mauro | Similar eicosapentaenoic acid and docosahexaenoic acid plasma levels achieved with fish oil or krill oil in a randomized double-blind four-week bioavailability study[END_REF] . These contradictory results may account for different experimental designs, especially different EPA/DHA ratios, and prevent accurate comparison. Moreover, the impact of the molecular lipid species form was often studied by using fish oil for TG and krill oil for PL. However, it is worth noting that krill oil only provides 40-60% of n-3 PUFA in the form of PL, while the remaining fraction originates from TG and free fatty acids (FFA) [START_REF] Castro-Gómez | Comprehensive study of the lipid classes of krill oil by fractionation and identification of triacylglycerols, diacylglycerols, and phospholipid molecular species by using UPLC/QToF-MS[END_REF][START_REF]Europea ood Safety uthor ty E S ) 9) Safety of 'l p d extract from Euphaus a superba' as a novel food ingredient[END_REF] . As a result, the role of PL as a lipid carrier on n-3 LC-PUFA metabolic fate is not clearly established. To our knowledge, only one study provided n-3 PUFA only as TG or as PL, and showed a better lymphatic absorption of n-3 LC-PUFA for rats fed PL vs TG [START_REF] Cansell | Marine lipid-based liposomes increase in vivo FA bioavailability[END_REF] . However, PL were structured in liposomes while TG were provided as fish oil in bulk phase, so that the physical state of the two formulations was not comparable.

Thereby, the objective of the present study is to provide data on the digestibility and the intestinal absorption of n-3 LC-PUFA according to their molecular lipid species, only in the bulk oily phase. Special attention was paid to ensure that the EPA/DHA ratio in the dietary lipid sources remained the same in order to ensure comparable results. n-3 LC-PUFA esterified in TG or PL were first subjected to an in vitro lipolysis in a titrimetric model. Intestinal absorption of n-3 LC-PUFA was studied in vivo in a rat lymph duct fistula model. The lipid and fatty acid compositions of the lymph were analyzed. It is worth noting that in vivo rodent models are considered as appropriate study models to assess oral compound bioavailability. They are often used in single dose protocols or supplementation studies [START_REF] Couëdelo | Impact of various emulsifiers on ALA bioavailability and chylomicron synthesis through changes in gastrointestinal lipolysis[END_REF][START_REF] Couëdelo | Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion[END_REF][START_REF] Michalski | Multiscale structures of lipids in foods as parameters affecting fatty acid bioavailability and lipid metabolism[END_REF][START_REF] Cansell | Marine lipid-based liposomes increase in vivo FA bioavailability[END_REF] . Indeed, similar physiological and physicochemical events, in particular those implying lipolytic enzymes, are described in both rodents and humans. Moreover, lymph analysis gives direct information on intestinal bioavailability, in contrast to blood, where the lipid composition may be contaminated by lipoproteins from liver metabolism. Thus, rat studies provide useful data to better understand the early stages of lipid digestion and investigate the intestinal bioavailability of FA of interest.

METHODS

Animals

Male Wistar rats (8 weeks-old, body weight: 300-350 g) were obtained from Elevage Janvier (Saint Genest sur l'Isle). All experiments were carried out in compliance with the ethic committee in

Experimental design

Twenty-four hours before surgery, rats were fed a fat free diet (SAFE) with free access to water.

Each rat was placed under anesthesia by an intra-peritoneal injection of a ketamine/xylazine mixture (100/10 mg/kg, respectively; Axience). A polyethylene catheter (0.95 mm x 15 cm; Biotrol) was inserted into the main mesenteric lymph duct as described by Bollman et al. [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF] and Couëdelo et al. [START_REF] Couëdelo | Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion[END_REF] . Immediately after surgery, rats were randomly assigned to one of the 3 experimental groups (6 rats/group), corresponding to one of the three n-3 LC-PUFA enriched formulations (TG n-3 , TG n-3 +PL veg , and TG veg +PL n-3 ). A volume, equivalent to 1 g of formulation, was orally administrated by gavage, providing respectively 8.6 and 22.3 mg of EPA and DHA per rat. Each rat was then placed in an individual restraining cage (rat compartment 228 mm x 89 mm), in a warm environment with freely available water. After gavage, lymph was collected during 6 h in a collection tube stored in ice. To prevent pain, rats received an intra-peritoneal injection of buprenorphine (0.02 mg/kg; Axience) 1 h before and 2 h after surgery. At 6 h post-intubation, rats were euthanized by an intra-peritoneal injection of sodium pentobarbital (Axience) and lidocaine (Ceva).

Lipid formulations

The lipid formulations were prepared at room temperature under nitrogen in order to prevent PUFA oxidation. Three marine formulations were designed to provide a similar amount of FA, but differed by the chemical structure of lipids. More precisely, EPA and DHA were exclusively esterified either on TG or on PL molecules (Table 1).

Marine PL were extracted from Lecimarin F50 (Novastell) by solvent fractionation using cold acetone. Briefly, 1 mL of chloroform/methanol (2/1, v/v) was added to 1 g of marine lecithin and maintained at room temperature until complete solubilisation. After addition of 20 mL of cold acetone (-20 °C), the sample was homogenized and then centrifuged (1050 g, 5 min, 4 °C; Sorvall ST-40R, Thermo Fisher Scientific). The supernatant, containing the neutral lipids, was removed and the pellet, containing the polar lipids was extracted twice with cold acetone and stored at -20 °C. Phospholipid classes of marine polar lipid extract and of soya lecithin were characterized by proton nuclear magnetic resonance ( 1 H NMR) according to Cansell et al. [START_REF] Bollman | Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat[END_REF] (Table 1). The glyceride structure of the marine oil mixture and of the marine PL was enzymatically determined according to methods described below. In the marine TG (oil mixture), EPA was mainly esterified in the sn-1/3 positions of marine TG (26% in the sn-2 position) whereas DHA was mainly located in the sn-2 position (50%). In marine PL, both EPA and DHA were essentially distributed in the sn-2 position (>74%) (see Supplementary data 1).

Lipid analysis

The total lipids were extracted from the lymph according to the procedure described by Folch et al. [START_REF] Castro-Gómez | A high-performance direct transmethylation method for total fatty acids assessment in biological and foodstuff samples[END_REF] . PL and TG fractions from the lipid extract were separated by thin layer chromatography (TLC, glass plates 20x20 cm pre-coated with silica gel 60H) using a solvent mixture composed of hexane/diethyl ether/formic acid (75/35/1, v/v/v). After vaporization of 2,7-dichlorofluorescein (2,7-DCF; Sigma-Aldrich) and visualization under UV-light, the spots corresponding to PL and TG were extracted from the silica gel by the addition of 2.5 mL of chloroform/methanol (2/1, v/v).

After homogenization and centrifugation of the scraped fractions (1050 g, 5 min, 20 °C), the organic phase containing lipids was collected. The extraction step was repeated by the addition of distilled water (100 µL) and chloroform/methanol (2/1, v/v, 2 mL) to the silica gel phase. Lipid extraction from the silica gel ended by the addition of 2 mL of methanol to the silica gel phase, homogenization and centrifugation. The overall organic phases were pooled and dried under nitrogen. Finally, 2,7-DCF was removed using 0.4 mL of a KCl solution (0.8% in distilled water w/v) and 2 mL of chloroform/methanol (2/1, v/v). The organic phase was washed twice by the addition of 0.8 mL of a mixture of chloroform/methanol/KCL 0.8% in distilled water (15/240/235, v/v/v). Samples were stored at -20 °C until analysis.

The FA composition of the lipid formulations, the samples from the in vitro lipolysis experiments and those collected from the lymph, i.e. total lipids, TG and PL fractions, were submitted to the (trans)methylation procedure adapted from Castro-Gomez [START_REF] Sehl | A critical assessment of transmethylation procedures for n-3 long-chain polyunsaturated fatty acid quantification of lipid classes[END_REF][START_REF] Wolff | Positional distribution of fatty acids in cardiolipin of mitochondria from 21-day-old rats[END_REF] . The resulting fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) analysis (TRACE GC, Thermo Fisher Scientific) equipped with a flame ionization detector (FID) and a split injector. A fused-silica capillary column (BPX 70, 60 m×0.25 mm i.d., 0.25 μm film; SGE) was used with hydrogen as a carrier gas (inlet pressure: 120 kPa). The split ratio was 1:33. The column temperature program was as follows: from 160 °C, the temperature increased to 180 °C at 1.3 °C/min, and was maintained for 65 minutes before increasing at 25 °C/min to 230 °C, temperature that was maintained for 15 minutes. The injector and detector were maintained at 250 °C and 280 °C, respectively. GC peaks were integrated using Chromquest software (Thermofinnigan). FA were quantified using an internal standard (1 mg/mL in chloroform/methanol (2/1, v/v)) added at 10% of the lipid weight before the (trans)methylation procedure. ) )

The glyceride structure of lymph PL was achieved by phospholipase A2 (PLA 2 ) hydrolysis according to the Wolff et al [START_REF] Carrière | The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals[END_REF] method, using PLA 2 from bee venom (600 -2400 U/g; 1U = 1 µEq FA/h; Sigma-Aldrich). Briefly, 150 µL of a PLA 2 solution (1 mg/mL; 3 mM CaCl 2 ) were added to 2 mg of extracted lymph PL and 1 mL of diethyl ether. Samples were incubated under stirring, at 30

°C for 1 h. The reaction was stopped by solvent evaporation under nitrogen. Hydrolysis products were separated by TLC using a solvent mixture of chloroform/methanol/distillated water (65/25/4, v/v/v). FFA were scraped off and transferred into a glass tube for methylation. The proportion of FA esterified in the sn-2 position of PL was calculated by considering the proportions of each FA released as FFA compared to those from the two positions of the PL, with the following equation:

) )

In vitro intestinal lipolysis of lipid formulations

The enzymatic lipolysis of the lipid formulations was assessed using an in vitro digestion model that mimics the physiological conditions of the intestinal lumen according to Carriere et al. [START_REF] Cansell | Phospholipid profiles of oleaginous pressed cakes using NMR and gas chromatography[END_REF] and Minekus et al. [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF] with slight modifications. The lipolysis was initiated by 5 mL of a 25% lipid emulsion stabilized by 1.4% (w/v) of methyl cellulose (Sigma-Aldrich) and dispersed in 15 mL of an assay solution containing 0.6% (w/v) of methyl cellulose, 150 mM NaCl (Thermo Fisher Scientific), 1.4 mM CaCl 2 (Sigma-Aldrich) and 20 mM sodium deoxycholate (Sigma-Aldrich). This mixture was stirred in a temperature-controlled reaction vessel at 37 °C and the pH was adjusted to

Characterization of lymph chylomicrons

The size of the lymph chylomicrons (CM) was measured for each rat group by dynamic light scattering using Vasco TM (Cordouan Technologies). Lymph samples were firstly diluted in a NaCl solution in water (0.9%, w/v). Experiments were performed considering a water refractive index of 1.331 and a viscosity of 0.888 cP. The real part and the imaginary part of particles were fixed at 1.47 and 0.01, respectively.

Statistical analysis

Data were expressed as mean values with standard deviation (mean ± SD). Intergroup comparisons were made on the basis of their respective mean. For the in vitro lipolysis experiment (n = 3), multiple comparisons were analyzed using a two-way ANOVA with two independent factors (lipid formulation and time of lipolysis) followed by a Bonferroni post-hoc test, using GraphPad Prism version 7. Data issued from the in vivo intestinal absorption experiment (n = 6) were compared by a one-way ANOVA test followed by a Tukey post-hoc test, using R version 3.5.1 (Rcommander package). P values lower than 0.05 were considered to be statistically significant.

RESULTS

In vitro digestion of n-3 LC-PUFA is modulated by their molecular lipid species of intake

Marine formulations (TG n-3 , TG n-3 +PL veg , TG veg +PL n-3 ) were subjected to in vitro lipolysis in a onestep static digestion model, with a focus on the duodenal lipolysis (Figure 1). Results revealed that all lipid formulations were well hydrolyzed during the time course of the lipolysis but to a different extent according to the formulation used (p time <0.001; p lipids =0.05 and p interaction <0.001; two-way ANOVA test). At the end of the lipolysis kinetics (60 minutes), the presence of soya lecithin in the marine formulation (TG n-3 +PL veg ) tended to inhibit the total lipolysis compared with TG n-3 (lipolysis level of 21.3% ± 4.7 and 14.5 ± 1.5, for TG n-3 and TG n-3 +PL veg , respectively). The lipolysis of TG n-3 +PL veg formulation was decreased by 47% when compared with the formulation free of n-3 LC-PUFA (lipolysis level of 14.5% ± 1.5 and 27.3% ± 1.9 for TG n-3 +PL veg and TG veg +PL veg , respectively; p<0.05). When n-3 LC-PUFA were esterified in marine PL (TG veg +PL n- Influence of the molecular lipid species of the dietary lipids on the intestinal absorption of n-3

LC-PUFA

Six hours after lipid ingestion, no adverse effect was observed consecutive to lipid formulation intubation. The total FA concentration in the rat lymph was similar for the three marine formulations (Table 2). The proportions of EPA and DHA in lymph total FA did not show statistical differences between all groups. Lymph TG and PL were separated and their FA compositions were analyzed separately (Table 3). The relative proportions of lymph TG and PL were similar regardless of the molecular n-3 LC-PUFA carrier (p>0.05). Thereby, TG was the major lipid fraction (71% of total FA) while PL only represented 7% of total FA. Lymph TG presented a similar FA profile between the 3 groups (p>0.05). Saturated FA (SFA) and monounsaturated FA (MUFA) were the two major FA classes and PUFA were mainly represented by n-6 PUFA. Considering n-3 PUFA, EPA and DHA levels in lymph TG were similar in the 3 groups. In lymph PL, the incorporation of n-3 LC-PUFA and specifically EPA was quite low. DHA levels tended to be higher in the TG veg +PL n-3 than in TG n-3 and TG n-3 +PL veg (p=0.10). The proportions of the main FA esterified in the sn-2 position of lymph lipids were defined (Table 4).

We noted that, SFA, MUFA and PUFA were equally distributed in the three positions of lymph TG for all groups. In lymph PL, SFA were mostly esterified in the sn-1 position and PUFA in the sn-2, irrespective of the groups. Considering n-3 LC-PUFA, EPA and DHA were mainly located in the sn-2 position of lymph PL regardless the molecular FA carrier (TG vs PL). DHA was equally esterified in the three positions of lymph TG when provided as marine TG (TG n-3 and TG n-3 +PL veg )

whereas it was mainly located in the sn-1/3 positions when ingested as marine PL (TG veg +PL n-3 ; p<0.001). More precisely, the intake of marine PL decreased by 46% the proportion of DHA esterified in the sn-2 position of lymph TG. However, EPA was mainly inserted in the sn-1/3 positions of lymph TG in all groups.

Characterization of lymph chylomicrons

Lymph chylomicrons were analyzed in terms of particle size. Data revealed that the mean diameter of CM were comparable between the 3 groups (223 ± 28, 248 ± 46 and 227 ± 47 nm for TG n-3 , TG n- 3 +PL veg and TG veg +PL n-3 , respectively).

DISCUSSION

This study aims at evaluating the impact of the molecular lipid species of dietary n-3 LC-PUFA on (i) their in vitro digestibility and (ii) their intestinal bioavailability, in rats fed marine TG or PL.

Only few studies tried to couple in vitro lipid hydrolysis with in vivo absorption and they dealt with plant lipids [START_REF] Couëdelo | Impact of various emulsifiers on ALA bioavailability and chylomicron synthesis through changes in gastrointestinal lipolysis[END_REF][START_REF] Couëdelo | Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion[END_REF] . A recent study used marine lipids [START_REF] Murota | Roe-derived phospholipid administration enhances lymphatic docosahexaenoic acid-containing phospholipid absorption in unanesthetized rats[END_REF] . However, the formulations did not have a similar EPA/DHA ratio and did not allow comparison of PL as n-3 carrier or as emulsifier. In our work, lipid formulations were designed to provide a similar amount of EPA and DHA, esterified exclusively either in TG or in PL molecules. Because PL display surfactant properties that may influence lipid digestibility and absorption, a formulation containing soya lecithin instead of marine PL (TG n-3 +PL veg ) was also assessed. Results showed that even if the in vitro lipolysis level was dependent on the type of the formulation used, EPA and DHA were similarly absorbed, in vivo, regardless of the lipid carrier. However, they were differently distributed in lymph lipid fractions.

Specifically, when provided as marine PL, LC-PUFA tended to be better incorporated in lymph PL and DHA was more externalized in the glycerol backbone of lymph TG. To our knowledge, this is the first study analyzing the molecular structure of lymph lipid fractions using marine TG and PL as dietary n-3 LC-PUFA carriers.

Gastro-intestinal digestion can be considered as a limiting step of bioavailability. This is because dietary lipids first have to be hydrolyzed in the gastro-intestinal tract, before being absorbed by the intestinal brush border. In our study, we did not perform a gastric phase before the duodenal phase, which could be a limitation. However, (1) duodenal lipolysis represents nearly 70% of total lipolysis [START_REF] Carriere | Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans[END_REF] and ( 2) the efficiency of the intestinal lipolysis is not called into question by the gastric phase. Our data confirmed that lipolysis extent depended on the affinity of pancreatic lipase toward the lipid species. The presence of n-3 LC-PUFA esterified in marine TG (TG n-3 +PL veg vs TG veg +PL veg ) dramatically impaired the lipolysis rate, due to the molecular structure of EPA and DHA which led to a steric hindrance effect in in vitro studies [START_REF] Akanbi | Pancreatic lipase selectively hydrolyses DPA over EPA and DHA due to location of double bonds in the fatty acid rather than regioselectivity[END_REF][START_REF] Bottino | Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis[END_REF] . In agreement with other studies [START_REF] Couëdelo | Lymphatic absorption of α-linolenic acid in rats fed flaxseed oil-based emulsion[END_REF][START_REF] Nishimukai | Enteral administration of soyabean lecithin enhanced lymphatic absorption of triacylglycerol in rats[END_REF] , we observed an inhibitory effect of soya PL (TG n-3 vs TG n-3 +PL veg ). This could be interpreted as an impediment of the binding of pancreatic lipase/colipase at the oil-water interface, decreasing TG availability for hydrolysis [START_REF] Borgström | Importance of phospholipids, pancreatic phospholipase A2, and fatty acid for the digestion of dietary fat: in vitro experiments with the porcine enzymes[END_REF][START_REF] Larsson | Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase[END_REF] . Moreover, the PL nature influenced the lipid hydrolysis level (TG veg +PL veg vs TG veg +PL n-3 ). In our experiment, the porcine pancreas extract contained PLA 2 . It is well known that PLA 2 activity is modulated by the nature of the polar head group of PL and to a lesser extent by the FA nature, i.e. chain length or unsaturation degree [START_REF] De Haas | Purification and properties of phospholipase A and its zymogen from porcine pancreas[END_REF][START_REF] Jain | Substrate specificity for interfacial catalysis by phospholipase A2 in the scooting mode[END_REF] .

Several studies using liposomes [START_REF] Ghomashchi | Interfacial catalysis by phospholipase A2: substrate specificity in vesicles[END_REF][START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF] and emulsion droplets [START_REF] Bahnson | Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer[END_REF] showed that PLA 2 would preferentially bond to anionic PL rather than zwitterionic PL. Thus, in our study, a part of the decreased lipolysis observed with TG veg +PL n-3 formulation could be attributed to the PL composition (5% of anionic PL in PL n-3 vs 39% in PL veg ).

The products generated by the lipid hydrolysis (FFA, 2-MG, and lysophospholipids (lysoPL)) are micellized by biliary salts before being absorbed through the intestinal brush border. In the enterocytes, the lipolysis products are re-synthesized in TG and PL, compacted into CM to be released into the lymph compartment before reaching the bloodstream. In our experiment, the CM diameter was similar, whatever the molecular carriers used, suggesting that the structural properties of CM were neither impacted by the presence of PL in the formulation, nor by the molecular n-3 LC-PUFA carrier used, nor by the variations of lipolysis rates observed in vitro. This finding is in accordance with a previous study demonstrating that the lipid composition of CM did not differ in rats fed fish oil or marine PL [START_REF] Cansell | Marine lipid-based liposomes increase in vivo FA bioavailability[END_REF] .

In all experimental groups, the FA quantification and FA profile of total lymph lipids were similar, suggesting that, under our test conditions, the digestion and the intestinal absorption of n-3 LC-PUFA were not modulated by the molecular n3 LC-PUFA carrier (TG n-3 vs PL n-3 ). Moreover, lymph lipids were mainly composed of PL and TG and the incorporation of EPA and DHA in lymph lipid fractions occurred independently of the molecular lipid species used (i.e. TG, PL, lysoPL, FFA) [START_REF] Cansell | Marine lipid-based liposomes increase in vivo FA bioavailability[END_REF][START_REF] Singer | Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2[END_REF][START_REF] Bahnson | Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer[END_REF][START_REF] Subbaiah | Enhanced incorporation of dietary DHA into lymph phospholipids by altering its molecular carrier[END_REF] . Indeed, the FA profile of lymph TG revealed similar n-3 PUFA levels between the three groups, suggesting that EPA and DHA were incorporated in lymph TG regardless of the molecular FA carrier. However, in lymph PL, DHA levels tended to increase when provided as marine PL (TG veg +PL n-3 ) compared with the TG form of intake (TG n-3 and TG n-3 +PL veg ). This data agrees with a previous study where the proportions of n-3 LC-PUFA in CM-PL were increased with marine PL compared with fish oil [START_REF] Cansell | Marine lipid-based liposomes increase in vivo FA bioavailability[END_REF][START_REF] Murota | Roe-derived phospholipid administration enhances lymphatic docosahexaenoic acid-containing phospholipid absorption in unanesthetized rats[END_REF] . It has been suggested that FA absorption could be lower in anesthetized rats than in conscious animals. Nonetheless, the relative distribution of DHA in lymph lipid fractions is expected to be similar regardless of the consciousness state of the rats [START_REF] Subbaiah | Enhanced incorporation of dietary DHA into lymph phospholipids by altering its molecular carrier[END_REF] .

The analysis of the glyceride structure of TG clearly showed that the molecular n-3 LC-PUFA carrier influenced the mechanisms involved in the re-synthesis of lymph lipids. In the enterocyte, lymph TG are mainly synthesized by the 2-MG pathway. We found that marine TG intake (TG n-3 and TG n-3 +PL veg ) led to an equimolar distribution of DHA in the three positions (33% of each) of lymph TG whereas the DHA provided was 56% esterified in the sn-2 position in marine TG formulation. This showed that the sn-2 position of the dietary TG was not fully conserved in lymph TG. A similar conclusion was drawn when structured TG rich in alpha-linolenic acid (ALA; 18:3 n-3) were used, i.e. only 50% of ALA located in the sn-2 position of dietary TG were recovered at this position in rat lymph TG [START_REF] Couëdelo | The fraction of α-linolenic acid present in the sn-2 position of structured triacylglycerols decreases in lymph chylomicrons and plasma triacylglycerols during the course of lipid absorption in Rats[END_REF] . This was interpreted as either (i) an isomerization of 2-MG into 1-MG prior to the lipolytic activity of pancreatic lipase [START_REF] Mattson | The digestion and absorption of triglycerides[END_REF] or (ii) a hydrolysis of the absorbed 2-MG by an intestinal lipase in the enterocyte [START_REF] Chon | Intestinal monoacylglycerol metabolism: developmental and nutritional regulation of monoacylglycerol lipase and monoacylglycerol acyltransferase[END_REF] . This suggested that new metabolic pathways could take place as soon as the concentration of a lipid species varied greatly, thus ensuri g a "homeostat c" state. On the contrary, in marine PL, DHA was exclusively (>74%) esterified in the sn-2 position of the glycerol backbone. After PL digestion, DHA was mainly released as FFA, due to a predominant activity of PLA 2 , specific of the sn-2 position of PL [START_REF] Cohn | Dietary phospholipids, hepatic lipid metabolism and cardiovascular disease[END_REF] . Thus, the presence of DHA in the sn-2 position of lymph TG suggested that the glycerol-3-phosphate (G3P) pathway was involved for the de novo TG synthesis using un-esterified DHA. Indeed, the G3P pathway could account for at least 15% of lymph TG synthesis. Nevertheless, the major pathway for re-synthesis of lymph TG seemed to be the 2-MG pathway, leading to the incorporation of a large part of free DHA on the 2-MG backbone. In this way, TG veg +PL n-3 ensured a DHA enrichment in the sn-1/3 positions of lymph TG compared with the marine TG groups (TG n-3 and TG n-3 +PL veg ).The analysis of the glyceride structure of PL showed the same n-3 LC-PUFA repartition in the three dietary groups. As lymph PL mainly originate from the re-acylation of 1-lysoPL, this suggested that, irrespective of the amount of free DHA in the enterocyte, the composition of lymph PL is highly regulated with an incorporation mainly in the sn-2 position.

These mechanisms are confirmed with the metabolic fate of EPA. Indeed, EPA was less esterified in the sn-2 position of lymph TG when provided as marine PL than as marine TG. Nevertheless, since EPA was mainly distributed in the sn-1/3 positions of marine TG and in sn-2 position of marine PL, it was essentially absorbed as FFA after gastro-intestinal hydrolysis. Free EPA was then incorporated either in the sn-1/3 positions of lymph TG or in the sn-2 position of lymph PL.

In conclusion, this study was undertaken to analyze the impact of different molecular carriers of n-3 LC-PUFA that could increase their bioavailability, based on in vivo data. Our results showed that dietary PL from vegetable or animal sources added to the oily formulations, at the ratio of 10%, did not increase lipid absorption. Although lipolysis differed according to the composition of the formulation, it seemed that the molecular carrier did not greatly modify the intestinal absorption of n-3 LC-PUFA. In contrast, the nature of the dietary lipids might modulate FA incorporation and distribution in lymph lipid fractions. However, it would be interesting to complete this study with the analysis of gene and protein expressions as well as with the activities of some enzymes implied in the intracellular lipid metabolism pathways to consolidate our data. The fact that, when provided as marine PL, DHA was better incorporated in lymph PL and was externalized in lymph TG, may further affect its metabolic fate and tissue accretion. Indeed, previous reports indicated that using PL as n-3 LC-PUFA carriers can be more efficient than TG regarding the metabolization and hypoadipogenic effect of EPA and DHA [START_REF] Cansell | Marine phospholipids as dietary carriers of long-chain polyunsaturated fatty acids[END_REF][START_REF] Graf | Age dependent incorporation of 14C-DHA into rat brain and body tissues after dosing various 14C-DHA-esters[END_REF][START_REF] Rossmeisl | Metabolic effects of n-3 PUFA as phospholipids are superior to triglycerides in mice fed a high-fat diet: possible role of endocannabinoids[END_REF][START_REF] Awada | n-3 PUFA added to high-fat diets affect differently adiposity and inflammation when carried by phospholipids or triacylglycerols in mice[END_REF][START_REF] Valenzuela | Effect of supplementation with docosahexaenoic acid ethyl ester and sn-2 docosahexaenyl monoacylglyceride on plasma and erythrocyte fatty acids in rats[END_REF](51) .  n-3 PUFA 3.9 ± 0.2 a 3.8 ± 0.1 a 3.7 ± 0.9 a 18:3 n-3 1.1 ± 0.1 a 0.7 ± 0.1 b 1.0 ± 0.2 a 20:5 n-3 0.5 ± 0.0 a 0.6 ± 0.0 a 0.5 ± 0.2 a 22:6 n-3 1.8 ± 0.1 a 2.0 ± 0.2 a 1.9 ± 0.

  Bordeaux and accepted by The French Ministry under the number n o 2017031014448864). Rats were treated in accordance with the European Communities Council Guidelines for the Care and Use of Laboratory Animals (2010/63/EU). All experiments conformed to the Guidelines for the Handling and Training of Laboratory Animals. Rats were housed 4 per cage (480 mm x 375 mm x 210 mm) for at least 7 days before the experiment in a controlled environment, at constant temperature (22 °C ± 1 °C) and humidity (60%) with free water and food access and a light/ dark cycle of 12 h/12 h.

  FFA 17:0 (Nu-Chek Prep) was used as an internal standard for the in vitro lipolysis study. TG 17:0 (Nu-Chek Prep) and PC 17:0 (Avanti Polar Lipids) were used for TG and PL quantification of the lymph samples, respectively.The proportion of FA esterified in the sn-2 position of lymph TG was determined using the stereospecificity of lipase. Lymph TG (3 mg) were mixed with 1 mL of lipase B Candida Antartica (>2000 U/g; 1U = 1µEq FA/h; Sigma-Aldrich) in solution in ethanol (4 mg/mL, w/v) and placed under stirring, at 30 °C for 3 h. Lipids were extracted 6 times by 10 mL of diethyl ether. Hydrolysis products were separated by TLC using a solvent mixture composed of hexane/diethyl ether/acetic acid (70/30/1, v/v/v). After vaporization of 2,7-DCF and visualization under UV-light, the spots corresponding to the 2-monoglycerides (2-MG) were scraped off and transferred into a glass tube for their transmethylation. The proportion of FA esterified in the sn-2 position of TG was calculated by considering the proportions of each FA from 2-MG compared to those from the three positions of the TG, with the following equation:

7. 5 .

 5 The lipolysis was initiated by adding 1 mL of porcine pancreas extract solution (6.7 mg/mL in distilled water, pH 7.5; Sigma-Aldrich) to the reaction vessel. The pH was kept constant at 7.5 for 60 min with 0.1 M NaOH (Chem-Lab) automatically controlled by a pH-stat device (pHstat Titrando, Metler Toledo). Samples (0.5 mL) were collected at times ranging from 0 to 60 min for lipid extraction and analysis. Lipid extraction was immediately performed by mixing each sample vigorously (0.5 mL) with 200 μL HCl (0.1 N; Thermo Fisher Scientific) and 5 mL chloroform/methanol (2/1, v/v) in a glass tube. After phase separation, the upper aqueous phase was removed and the lower phase containing the total lipids was dried under nitrogen, dissolved in 3 mL chloroform/methanol (2/1, v/v), and stored at -20 °C until analysis. FFA, from TG and PL lipolysis, were isolated from other lipid fractions by TLC (glass plates 20x20 cm pre-coated with silica gel 60H (Merck KGaA)) using a solvent mixture composed of hexane/diethyl ether/acetic acid (80/20/1, v/v/v). Spots were visualized under UV-light after vaporization of 2,7-DCF in ethanol solution (0.2%, w/v). FFA were scraped off and transferred into a glass tube for methylation and GC analysis. At each time point of the kinetics (0, 5, 10, 15, 30 and 60 min), the hydrolysis level (% FFA/total FA) was calculated. A deprived n-3 LC-PUFA formulation composed of 10

Figure 1 :

 1 Figure 1: Effect of lipid formulation on lipolysis levels using an in vitro digestion model. TG n-3 ( • ), TG n-3 +PL veg ( ▲ ), TG Veg +PL n-3 () and TG veg +PL veg (). Lipolysis levels are expressed as % FFA versus total acyl chains in the residual triglycerides, diglycerides, monoglycerides and FFA. Values are means ± SD (n = 3).Values with different superscript letters (a, b) for the same time are significantly different at p<0.05, two-way ANOVA followed by Bonferroni post-hoc test.

3 :

 3 oil phase with n-3 LC-PUFA esterified in triglycerides; TG n-3 +PL veg : oil and soya lecithin mix with n-3 LC-PUFA esterified in triglycerides; TG veg +PL n-3 : oil and marine PL mix with n-3 LC-PUFA esterified in phospholipids.[START_REF] Dyall | Neurological benefits of omega-3 fatty acids[END_REF] Values are expressed as mean ± SD, n=6.Values with different superscript letters (a, b) in the same line are significantly different at p<0.05, ANOVA followed by Tukey post-hoc test.TG: triglycerides, PL: phospholipids, SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids.

3 :

 3 oil phase with n-3 LC-PUFA esterified in triglycerides; TG n-3 +PL veg : oil and soya lecithin mix with n-3 LC-PUFA esterified in triglycerides; TG veg +PL n-3 : oil and marine PL mix with n-3 LC-PUFA esterified in phospholipids.[START_REF] Dyall | Neurological benefits of omega-3 fatty acids[END_REF] Values are expressed as mean ± SD, n=6.Values with different superscript letters (a, b) in the same line are significantly different, p>0.05, ANOVA followed by Tukey post-hoc test.TG: triglycerides, PL: phospholipids, SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids.

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	https://doi.org/10.1017/S0007114519001491

  Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 11 Aug 2019 at 16:06:29, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.

  Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 11 Aug 2019 at 16:06:29, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114519001491 experiments were performed in triplicate, for each formulation, i.e. TG n-3 , TG n-3 +PL veg , and TG veg +PL n-3 .

wt.% of soya lecithin dissolved in a vegetable oil mixture (copra/palm/oleic sunflower oil; 6/30/63, w/w/w; TG veg +PL veg ) was also assessed as a control (see Supplementary data 2). Lipolysis

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https://doi.org/10.1017/S0007114519001491

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Table 1 :

 1 Fatty acid and phospholipid compositions of the three experimental lipid formulations TG n-3 : oil phase with n-3 LC-PUFA esterified in triglycerides; TG n-3 +PL veg : oil and soya lecithin mix with n-3 LC-PUFA esterified in triglycerides; TG veg +PL n-3 : oil and marine PL mix with n-3

		TG n-3	1	TG n-3 +PL veg	TG veg +PL n-3
				g/100g of total fatty acids
	 SFA	32.6		31.4	31.0
	16:0	15.9		15.4	15.4
	18:0	3.5		3.4	3.3
	 MUFA	55.0		57.0	56.7
	18:1 n-9	52.8		54.6	55.1
	 PUFA	12.4		11.6	12.3
	 n-6 PUFA	8.6		7.7	8.7
	18:2 n-6	8.2		7.2	8.4
	20:4 n-6	0.2		0.2	0.0
	 n-3 PUFA	3.8		3.9	3.4
	18:3 n-3	0.7		0.4	0.7
	20:5 n-3	0.8		0.9	0.7
	22:6 n-3	2.0		2.2	1.9
	n-6/n-3	2.3		2.0	2.6
				g/100g of total phospholipids
	Phosphatidylcholine			31.7	73.8
	Phosphatidylinositol			29.3	3.5
	Phosphatidylethanolamine			29.6	5.2
	Phosphatidylserine			5.4	-
	Phosphatidic acid			4.0	1.2
	Sphingomyelin			-	2.9

[START_REF] Riediger | A systemic review of the roles of n-3 fatty acids in health and disease[END_REF]

Table 2 :

 2 Effect of the molecular carrier form of n-3 LC-PUFA on fatty acid composition of total lipids of lymph rats, 6 h after intubation

		TG n-3	1	TG n-3 +PL veg	TG veg +PL n-3
				g/100g of total fatty acids
	 SFA	27.4 ± 1.5 a	26.4 ± 1.0 a	26.3 ± 1.6 a
	16:0	17.8 ± 1.0 a	18.5 ± 0.7 a	17.9 ± 1.0 a
	18:0	4.8 ± 0.4 a	5.1 ± 0.8 a	5.0 ± 0.3 a
	 MUFA	49.9 ± 3.5 a	52.4 ± 3.1 a	52.5 ± 3.8 a
	18:1 n-9	45.5 ± 4.6 a	48.0 ± 3.5 a	48.8 ± 4.3 a
	 PUFA	22.8 ± 2.8 a	21.2 ± 2.1 a	21.3 ± 3.0 a
	 n-6 PUFA	18.8 ± 2.6 a	17.3 ± 2.0 a	17.5 ± 2.5 a
	18:2 n-6	14.2 ± 2.0 a	12.7 ± 1.3 a	13.5 ± 2.0 a
	20:4 n-6	3.8 ± 0.7 a	3.8 ± 0.9 a	3.5 ± 0.7 a

Table 3 :

 3 Effect of the molecular carrier form of n-3 LC-PUFA on fatty acid composition of triglycerides and phospholipids of lymph rats, 6 h after intubation

				Lymph triglycerides	Lymph phospholipids
		TG n-3	1	TG n-3 +PL veg TG veg +PL n-3	TG n-3	TG n-3 +PL veg TG veg +PL n-3
					g/100g of total fatty acids
	 SFA	26.3 ± 2.1 2a 25.9 ± 1.9 a	25.7 ± 1.9 a	39.5 ± 1.1 a	38.4 ± 1.2 a	39.2 ± 1.3 a
	16:0	17.8 ± 1.0 a	18.4 ± 0.9 a	17.7 ± 1.0 a	20.4 ± 1.2 a	18.8 ± 0.8 b	19.9 ± 1.2 ab
	18:0	4.0 ± 0.4 a	4.1 ± 0.5 a	4.0 ± 0.3 a	17.6 ± 1.1 a	18.3 ± 1.3 a	17.8 ± 1.1 a
	 MUFA	53.6 ± 2.9 a	55.7 ± 3.4 a	55.9 ± 3.8 a	13.7 ± 2.6 a	13.4 ± 1.9 a	14.5 ± 3.2 a
	18:1 n-9	49.0 ± 4.1 a	51.3 ± 3.8 a	52.3 ± 4.0 a	9.1 ± 2.8 a	8.7 ± 2.0 a	8.7 ± 3.5 a
	 PUFA	20.1 ± 2.8 a	18.4 ± 1.7 a	18.6 ± 2.1 a	46.9 ± 2.0 a	48.2 ± 2.5 a	46.3 ± 2.4 a
	 n-6 PUFA 16.2 ± 2.4 a	14.6 ± 1.7 a	14.9 ± 1.6 a	43.7 ± 2.2 a	44.8 ± 2.4 a	42.3 ± 2.1 a
	18:2 n-6	13.2 ± 2.1 a	11.7 ± 1.4 a	12.5 ± 1.5 a	23.9 ± 2.0 a	24.6 ± 1.7 a	24.2 ± 2.3 a
	20:4 n-6	2.3 ± 0.4 a	2.3 ± 0.6 a	2.0 ± 0.4 a	18.2 ± 1.5 a	18.8 ± 2.3 a	16.9 ± 1.9 a
	 n-3 PUFA	3.8 ± 0.4 a	3.7 ± 0.1 a	3.5 ± 0.9 a	3.1 ± 0.2 a	3.3 ± 0.2 a	3.9 ± 1.1 a
	18:3 n-3	1.1 ± 0.2 a	0.7 ± 0.1 b	1.0 ± 0.1 a	0.3 ± 0.0 a	0.3 ± 0.0 b	0.3 ± 0.0 a

Table 4 :

 4 Effect of the molecular carrier form of n-3 LC-PUFA on fatty acid distribution in triglycerides and phospholipids of lymph rats, 6 h after intubation

			Lymph triglycerides		Lymph phospholipids
	TG n-3	1	TG n-3 +PL veg TG veg +PL n-3	TG n-3	TG n-3 +PL veg TG veg +PL n-3



% esterified in the internal sn-2 position

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), the lipolysis seemed to be slower at the beginning of the kinetics compared with the control curve (TG veg +PL veg ), however at 60 min, the lipolysis levels were not statistically different (21.5% ±

[START_REF] Saini | Omega-3 and omega-6 polyunsaturated fatty acids: Dietary sources, metabolism, and significance -A review[END_REF].3 and 27.3% ± 1.9 for TG veg +PL n-3 and TG veg +PL veg , respectively). Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 11 Aug 2019 at 16:06:29, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114519001491

ACKNOWLEDGEMENTS

The study was partly financially supported by the French National Association for Research and Technology (ANRT, N° 2015/1182). Authors are grateful to Novastell and in particular Thierry Coste for providing Lecimarin F50, and also to Polaris for supplying fish oils. The authors also wish to thank Axelle Grélard from Bordeaux University, CNRS, CBMN-UMR 5248, for RMN analysis.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.


H Vahabi

R Sonnier

B Otazaghine

G Le Saout

J M Lopez-Cuesta
email: marie.lopez-cuesta@mines-ales.fr

Keywords: mineral filler, nanocomposite, halloysite, sepiolite, flame retardancy. NANOKOMPOZYTY

Nanocomposites of polypropylene/polyamide 6 blends based on three different nanoclays: thermal stability and flame retardancy Summary -The influence of various layered silicates: sepiolite (needle-like structure), halloysite (nanotube structure) or organomodified montmorillonite (lamellar structure) in combination with phosphorous flame retardants [ammonium polyphosphate (APP) and aluminum diethylphosphinate (OP)] on the properties and morphologies of compatibilized PP/PA 6 blends has been investigated. Thermal degradation and fire retardancy of these blends were explored using TGA, cone calorimeter, pyrolysis flow combustion calorimeter (PCFC). Morphology of nanocomposites and residues as well of the chemical structure formed after cone calorimeter tests were investigated. The sepiolite/APP composition led to the better fire performance through the formation of a large amount of charred and expanded residue reinforced by the sepiolite fibres. The better compactness of this residue than that of the montmorillonite/APP one enables to account for the interest of sepiolite, whereas montmorillonite exerts the better fire retardant role, alone in the polymer blend.

INTRODUCTION

Since the last decade, the use of nanoparticles in polymers in order to improve their fire reaction has become an attractive field of research. Different parameters, such as chemical composition, microstructure, specific surface area, shape, particle size distribution, surface modification as well as their dispersion state in the matrix govern their contribution to flame retardancy [START_REF] Jiang | Polymer Nanocomposites" Chap. 11[END_REF][START_REF] Laoutid | [END_REF]. Among nanoparticles incorporated in polymers, organomodified layered silicates (OMLS) are of prime interest due to their availability, cost and contribution to flame retardancy [3]. Combinations of organomodified montmorillonites (OMMT) with various flame retardants, and particularly phosphorous compounds have attracted special attention [START_REF] Laoutid | Multi-components FR systems: polymer nanocomposites combined with additional materials" Chap. 12[END_REF] regarding the improvement of fire performance achieved for various polymers. Different mechanisms have been suggested to explain the effect of OMMT on flame retardancy as well as the interactions with phosphorous compounds acting as components of intumescent flame retardant (IFR) compositions [START_REF] Tang | [END_REF]6]. Hence, it appears likely that these interesting results will prompt researchers to investigate new combinations of layered silicates with phosphorous compounds. This paper focuses particularly on the comparison between three kinds of layered silicates presenting different morphologies, as well as their combinations with phosphorous compounds to improve the fire reaction of polypropylene/polyamide 6 blends in which polypropylene is the main component. PP/PA 6 blends have been used in the industry (e.g. ORGALLOYS from ARKEMA) since many years. Various solutions aiming to improve the fire retardancy of these blends which contains also compatibilizing agents can be considered. The interest of IFR systems has yet been proved for polyolefins and IFR systems containing PA 6 as char promoter have been proposed [7]. Moreover, it has been shown that IFR systems containing ammonium polyphosphate (APP) and PA 6 could generate synergistic effects on flame retardancy when combined with organomodified montmorillonites [8][9][10]. Nevertheless, even if the interest of modified sepiolite in combination with other kind of IFR (pentaerythritol instead of PA 6) in PP has been highlighted by Huang et al. [11], no investigation has been made about a comparison between various layered silicates having different microstructures, combined with IFR systems in PP. It had been shown in some cases that lamellar microstructures could be superior to fibrous, acicular, or tubular morphologies [6], due to their potential ability to build a tile-like structure at the surface of polymer when exposed to a heat source leading to its combustion. Nevertheless, other phenomena have to be taken into account such as:

-catalytic effects of silicate surface, particularly when surface modified, -influence on the morphology of the charred structure formed at the surface of material residue, -possible reactions occurring at high temperatures between the layered silicates and the phosphorous FR as it has been shown with nanooxides [12].

In this work, comparisons have been made with the three types of layered silicates alone in a PP/PA 6 compatibilized blend and also in combinations with the two phosphorous FR. The organomodified montmorillonite has been selected to enable a good dispersion in the PP phase through the use of SEBS-g-MA (SGM) which plays also the role of compatibilizing agent in the PP/PA 6 blend. Non-modified sepiolite and halloysite have been used. This last kind of layered silicate has also been selected since some authors have shown that it could exert a flame retardant action in various polymers [13].

EXPERIMENTAL

Materials

Polypropylene (PP) (SABIC ® -576P) and polyamide 6 (PA 6) (Rhodia C216-Technyl ® ) were used as polymer blend components with the following constant ratio PP/PA 6 80:20. SEBS-g-MA (Kraton FG 1901-named as SGM) was used as blend compatibilizer. Halloysite (named as HNT) was provided by Imerys Tableware New Zealand Ltd. Pangel ® -S9 Sepiolite (named as Sep.) from Tolsa (Spain) was supplied by Lavollée S.A. (France). An organomodified montmorillonite, modified with a quaternary ammonium (dimethyldihydrogenated tallow alkyl quaternary ammonium salt, named as N5) was provided by Southern Clay Products (Nanofil ® 5). The following phosphorous flame retardants supplied by Clariant were used as powders: an ammonium polyphosphate (AP 412 named as APP) and an organic aluminium phosphinate based composition containing a nitrogen compound acting as synergistic agent (Exolit OP1311).

Samples preparation

All blends were prepared in one step using a co-rotating twin screw extruder (Clextral BC 21, screw speed = 250 rpm, melt zone temperature = 180-240 °C). All nanoparticles were introduced in the molten polymer blend. The pellets were injection molded using a Krauss Maffei 50T apparatus (T = 200-240 °C, mold temperature = 80 °C) in order to obtain square sheet specimens 100 × 100 × 4 (mm 3 ). The sample names and compositions are given in Table 1 and Table 2. Prior to the processing, PA 6 and SEBS-g-MA were dried at 80 °C in a vacuum oven for 12 hours. Sepiolite, Nanofil ® 5 and Halloysite were also dried at 350 °C, 110 °C and 250 °C, respectively, to remove water physically bound.

T a b l e 1. Sample names and compositions

Methods of testing

-Thermogravimetric analysis (TGA) data were collected with a PerkinElmer Pyris-1 TGA instrument, under nitrogen, at 10 °C/min, from 50 °C to 900 °C.

-Evaluation of the flammability properties was made using Pyrolysis Combustion Flow Calorimeter (PCFC) and Cone Calorimeter devices manufactured by Fire Test Technology (FTT). For PCFC tests, the samples (1-4 mg) are heated at 1 °C/s from 20 °C to 750 °C in a pyrolyzer and the degradation products are transported by an inert flux, and then mixed with oxygen before entering a combustor at 900 °C where the decomposition products are completely burnt. Heat release rate (HRR) is measured as function of temperature according to oxygen depletion method (13.1 MJ of energy is released when 1 kg of oxygen is used according to Huggett's relation). Cone calorimeter tests were carried out on the square sheet specimens using an incident heat flux of 35 kW/m 2 , according to ISO 5660-1 standard.

-All images were obtained using a scanning electron microscopy (FEI Quanta 200 SEM) under high vacuum at a voltage of 15.0 kV with a spot size of 3 mm and a working distance of 8.2 mm (all samples were cryo-fractured).

-X-ray diffraction (XRD) data were collected using a BRUKER Advance D8 diffractometer in a q-q configuration employing the CuKa radiation (l = 1.54 A o ) with a fixed divergence slit size 0.6°and a rotating sample stage. The samples were scanned between 2.5°and 50°with the VANTEC-1 detector. The qualitative analysis was performed with the X'Pert High Score Plus software (version 2.1).

RESULTS AND DISCUSSION

Microstructures and properties of silicate layer nanocomposites

The first part of this work was devoted to the influence of three different geometries of layered silicates [sepiolite (needle-like microstructure), halloysite (nanotube microstructure) or organomodified montmorillonite (lamellar microstructure)] on flammability of PP/PA/SEBS-g-MA blend. The formulation was carried out without the phosphorus FR in order to evaluate the influence of different geometries of these clays. The microstructures of the layered silicates in the PP/PA/SGM blend are shown in Fig. 1.

It can be observed that all the silicates are dispersed in the blend even if some aggregates can be noticed. Consequently, it can be considered that nanostructures have been achieved. The presence of the organomodifier in Nanofil ® 5 seems not to modify the size of PA 6 nodules in the PP matrix. In order to investigate the exfoliation of montmorillonite in the matrix, XRD experiments have been performed (Fig. 2). In the N5 sample, the intercalation agent (dimethyl-distearyl-ammonium chloride) expands the interlayer spacing d001 of the montmorillonite up to 2.5 nm. We can notice a swelling of the phase (d001 = 2.8 nm) when the sample is heated up to 220 °C (temperature of the process). On preparing the PP/PA 6 blends sample, an increase of the basal spacing to 3.1 nm is observed. No complete montmorillonite exfoliation has taken place as previously reported in polymer blends based on polyethylene and N5 [START_REF] Merinska | Mechanical and barrier properties of PE and EVA/clay nanocomposites[END_REF].

TGA curves are presented in Fig. 3. While HNT and Sep compositions decomposes similarly to the polymer blend up to 480 °C, and show the presence of a residue af- ter this temperature, Nanofil ® 5 composition decomposes at lower temperature (close to 350 °C) but appears more stable than all other compositions from 450 °C. Moreover, a final residue close to 5 wt. % is obtained for all compositions. It can be noticed that residue of Nanofil ® 5 composition contains a little char fraction since this organomodified layered silicate contains 30 wt. % of organic fraction. The heat release (HRR) curves of the nanocomposites at cone calorimeter are presented in Fig. 4. These results clearly showed that Nanofil ® 5 (platelet geometry) significantly decreases the peak of HRR (around 50 %) compared to Sep or HNT, which seems to have no effect on pHRR reduction at this percentage of incorporation.

T a b l e 2. Sample names and compositions

Moreover, the total heat released (THR) at 500 °C appears slightly lower for Nanofil ® 5 composition, in comparison with this of the unfilled polymer blend (Table 3). However, it is well known that generally the incorporation of nanoparticles allows HRR values to be reduced but not the THR [START_REF] Gilman | Flame retardant mechanism of polymer-clay nanocomposites[END_REF][START_REF] Zanetti | [END_REF][17]. Time to ignition (TTI) is not modified by the incorporation of Nanofil ® 5 and sepiolite, but it is increased of 20 % with halloysite. It can be suggested that the tubular structure of halloysite may increase the thermal diffusivity through the sample and thereby delay ignition. It is also recognized that there is no general rule about this parameter for the incorporation of nanoparticles in polymers. These results concerning HRR values is in accordance with previous research works that showed the excellent performance of organomodified clays regarding reduction of pHRR, because of its effectiveness to build a protective mineral barrier at the surface of remaining material, hindering by a physical effect, the diffusion of volatile combustible from the filled blend and oxygen through it [START_REF] Gilman | Flame retardant mechanism of polymer-clay nanocomposites[END_REF][START_REF] Zanetti | [END_REF]. The formation of a mineral barrier in case of organomodified montmorillonite after polymer combustion results from polymer ablation but also from the migration of the dispersed clay platelets towards the surface exposed to heat source [18]. Such kind of mechanism requires exfoliated or intercalated microstructures and was only highlighted for this type of nanoparticles and not for other kind of silicates having different aspect ratio and different surface chemistry. Moreover, after thermal degradation of the organomodifier, montmorillonite exhibits an acidic surface, leading to enhanced catalytic activity, which promotes the formation of a charred structure reinforced by the clay platelets. The formation of this kind of barrier is confirmed here by the evolution of sample weight during the test (Fig. 5). The weight loss is slower for the composition with Nanofil ® 5 and the final residue at 800 s is significantly higher than the theoretical weight loss, taking into account the organic fraction (around 30 wt. %) of the organomodified montmorillonite. Conversely, for the other layered silicates, the weight of residue is close to this of the nanoparticle fraction in the polymer blend. Hence, halloysite and sepiolite seem not to exhibit any catalytic activity on the polymer blend degradation. It has to remind that in the various papers handling with the use of sepiolite and halloysite to exert a flame retardant action, their per-centage of incorporation is quite always much higher than 20 wt. %.

In the case of the combined presence of layered silicates and phosphorous compounds, the formation of new compounds could modify the structure of the protective layer formed in presence of organomodified montmorillonite. Moreover, it can be expected that a protective layer could also be formed from the reaction of other kind of silicates with phosphorous compounds.

Microstructures and properties of multicomponent compositions

The SEM microstructures of the PP/PA/SGM/APP, PP/PA/SGM/OP and the six multicomponent composi- 4.

At first, it can be noticed that phosphorous flame retardants (FR) impair the thermal stability of polymer blend (Fig. 7). The effect is more important with APP since it is less thermally stable than OP [12,19]. It can be also ascribed to possible reactions with PA 6, since PA 6 can play a role as charring agent in intumescent systems based on APP [START_REF] Tang | [END_REF].

T a b l e 4. TGA parameters of all samples under nitrogen

Sample code

T Two decomposition stages can be noticed in the TGA curve for the samples containing the layered silicates, particularly in the case of APP. The first stage ranges from 300 °C to 400 °C and can be ascribed to the phosphorous compound decomposition while the second one ranges from around 400 °C to 520 °C and seems to correspond to the decomposition of PP and PA 6.

In all cases, the onset temperature (T onset ) is lower than this of the compatibilized blend. APP/N5 composition presents the lower onset temperature of the multicomponent compositions due to the degradation of the dimethyl dihydrogenated tallow alkyl quaternary ammonium salt.

APP/HNT composition presents the highest thermal stability from 450 °C (see T 50 % in Table 4), but except for this composition, all the blends containing OP exhibit a better thermal stability than these containing APP.

For all compositions containing a phosphorous FR, significant amounts of residue are formed after polymer degradation, particularly for sample containing APP. Moreover, taken into account the respective percentage of nanoparticles and phosphorous FRs, a synergistic effect on residue formation can be highlighted for all compositions, except this containing OP and halloysite, taking into account the organic fraction of N5.

Pyrolysis combustion flow calorimetry has proved to be an advantageous technique to investigate fire behavior on very small specimens. Moreover, it has been highlighted in the literature that PCFC is more adapted to study chemical decomposition processes than physical ones (mass or heat transfer barrier effects) taking place in the condensed phase [10][11][12][13][START_REF] Merinska | Mechanical and barrier properties of PE and EVA/clay nanocomposites[END_REF][START_REF] Gilman | Flame retardant mechanism of polymer-clay nanocomposites[END_REF][START_REF] Zanetti | [END_REF][17][18][19][20][21][22].

The heat release rate (HRR) curves obtained from PCFC test are shown in Figs. 10 to 12 and the main parameters summarized in Table 5.

It appears that the presence of the SEBS-g-MA as compatibilizer in the PP/PA 6 blend tends to increase strongly the SumHRC (Heat Release Capacity). The incorporation of phosphorous compounds leads to a significant decrease of this value, particularly for OP. All APP compositions exhibit two peaks and the first one can be attributed to a first degradation stage of the polymer blend caused by reactions of APP with PA 6. For each kind of phosphorous compound, its combined incorporation with layered silicates leads to a strong reduction in SumHRC, apart from the APP/N5 composition which seems to cause a decomposition of the polymer blend through a catalytic effect, in a lower temperature range in comparison with all other compositions. In fact, it could be noticed that APP/N5 composition shows the lowest T 10 % for all the compositions containing APP.

The highest reduction in SumHRC for multicomponent compositions in comparison with ones containing only phosphorous compounds is achieved with sepiolite (respectively 16 and 17 % for APP and OP). Cone calorimeter curves (Fig. 13, Fig. 14) and data (Table 6) confirm the interest of the combinations between the phosphorous compounds and sepiolite. The lowest pHRR value is obtained for the APP/Sep composition. In addition, OP/Sep exhibits a pHRR value very close to this of OP/N5 which presents the lowest value for the OP compositions. On the whole, the compositions with APP leads to a better fire reaction than OP ones. Times to ignition (TTI) appear slightly lower for APP compositions, THR values decrease for all compositions in comparison with this of the compatibilized blend. Lower THR values are observed for APP/Sep and APP/N5. In addition, it can be noticed that for OP compositions, the combination with nanoparticles does not enable to reduce the THR.

Finally, from the cone calorimeter experiments, it appears that sepiolite shows the better results, then modified montmorillonite, and finally halloysite, which present only a limited interest. So, despite its ability to promote char formation at moderate ramp temperature as for TGA, APP/HNT combination lead only to a limited mass of residue in comparison with APP/N5 and above all APP/Sep, in case of a strong temperature rise such as in cone calorimeter experiments. Fig. 15 shows the significant differences in mass loss between the three APP-based compositions containing the layered silicates.

A comparison between PCFC and calorimeter data enables to account for the creation of a barrier effect through the formation of a protective layer. It was assumed that the barrier effect is only active in cone calorimeter test and negligible in PCFC due to the small sample size. Thus, it is supposed that the decrease of pHRR in cone calorimeter test (because of the incorporation of FR) should be higher (or at least equal to) than the decrease of pHRR {or Heat Release Capacity (HRC), [HRC is equal to the peak heat release rate (pHRR) divided by the heating rate]} in PCFC [23].

Here, the ratio between the HRC value of the flame retarded polymer sample in PCFC test [P-FR (PCFC)] and the HRC value of the non retarded polymer [P (PCFC)] is named R1. (In this study, HRC is equal to pHRR due to presence of only one major peak of HRR and a heating rate of 1 °C/s).

Fig. 13. Heat release rate curves of APP compositions at cone calorimeter test

The ratio between the pHRR of the flame retarded polymer sample in cone calorimeter test [P-FR (cone calorimeter)] and the pHRR of the non retarded polymer [P (cone calorimeter)] is named R2.

Figure 16 shows the representation of calculated R2 values (X-axis) versus calculated R1 values (Y-axis). For all FR samples, the points are plotted above the dotted line R1 = R2 (this line corresponds to a similar decrease in pHRR at cone calorimeter and at PCFC).

The extent of the discrepancies between experimental values and the dotted line R1 = R2 indicates that the barrier effect plays an important role in flame retardancy of these samples. The effect is particularly emphasized in the PP/PA/SGM/APP/Sep system. Figures 17 and18 shows, respectively, digital photographs and SEM micrographs of the surface of residues for compositions after cone calorimeter test. For all compositions, char structures showing an intumescent character have been formed. From a comparison of the PP/PA/SGM/APP/Sep and PP/PA/SGM/APP/N5 residues, it appears that the char is more porous for the second composition. This porosity may have major consequences on flame retardancy of these samples, since a porous char structure, thus permeable to gases (oxygen and volatile combustible from the sample), could not provide the best performance as barrier effect to protect the underlying material against the flame.

Thus, these pores could accelerate the evolution of gases to feed the spread of flame as well as the pyrolysis of the char and emission of airborne particles. The difference of the total smoke release between these samples is expressed in Fig. 19. The sample with a porous surface (PP/PA/SGM/APP/N5) evolves more quantity of smoke during cone calorimeter test.

The formation of compact charred and expanded structures for the residues seems due to the action of intumescent FR systems which improves the fire reaction of the PP which is the main component of the polymer blend matrix. The main components of these FR systems are the phosphorous compounds acting as acid sources and the PA 6 which acts as a carbon supplier. It has to be noticed that APP appears more effective than OP for almost all compositions. This can be ascribed to the formation of the charred structure at the first stages of PP decomposition, as it can be noticed on TGA and PCFC curves. The thermal stability of the charred structures formed at cone calorimeter which governs the final quantity of residue could depend on their compact character, according to Huang et al. [10]. In addition, it can be considered that the advantage of sepiolite in comparison with other nanoparticles tested could lie in its ability to reinforce mechanically this charred structure at nanometric level and thereby to limit its porosity. It can also be suggested that this enhancement of the quantity of char formed enable more phosphorus to act in the condensed phase, also leading to its reinforcement. Further investigations could concern the evaluation of the mechanical properties of the charred structures formed, the possible formation of new compounds formed by the reaction between the nanoparticles and the phosphorous FRs as well as the phosphorous fraction remaining in the residue.

CONCLUSIONS

Combinations of nanometric layered silicates such as organomodified montmorillonite and sepiolite with phosphorous FR such as ammonium polyphosphate and aluminum phosphinate allows the fire performances of a compatibilized PP/PA polymer blend to be improved. Conversely, the interest of halloysite, which present a nanotube structure is limited. Although organomodified montmorillonite led to the better performances when the nanoparticles were used without phosphorous FR in the polymer blend, the higher reduction in pHRR, THR, smoke release and the largest residue at cone calorimeter test has been obtained for a sepiolite/ammonium polyphosphate combination. The barrier effect created by the use of sepiolite has been highlighted by a comparison between cone calorimeter and PCFC data. This effect is ascribed to the formation of a very compact charred protective structure, possibly reinforced by the sepiolite fibres, and less porous than this formed with montmorillonite.

Fig. 1 .

 1 Fig. 1. SEM micrographs of nanocomposites: a) PP/PA/SGM/Sep, b) PP/PA/SGM/HNT, c) PP/PA/SGM/N5

T a b l e 3 .

 3 Cone calorimeter data for PP/PA/SGM polymer blends and its nanocomposites Composition TTI, s pHRR, kW/m 2

Fig. 7 .Fig. 6 .Fig. 5 .

 765 Fig. 7. TGA curves in nitrogen (10 °C/min) of PP/PA/SGM, PP/PA/SGM/APP and PP/PA/SGM/OP compositions

T a b l e 6 .

 6 Parameters of cone calorimeter test for all samples containing

Fig. 10 .Fig. 11 .Fig. 12 .

 101112 Fig. 10. Heat release rate curves of phosphorous FR compositions at PCFC test

Fig. 17 .Fig. 18 .

 1718 Fig. 17. Digital photographs of residues after cone calorimeter test: a) PP/PA/SGM/APP, b) PP/PA/SGM/OP, c) PP/PA/SGM/ APP/Sep, d) PP/PA/SGM/OP/Sep, e) PP/PA/SGM/APP/HNT, f) PP/PA/SGM/OP/HNT, g) PP/PA/SGM/APP/N5, h) PP/PA/ SGM/OP/N5

Fig. 19 .

 19 Fig. 19. Total smoke release for PP/PA/SGM/APP/Sep and PP/PA/SGM/APP/N5 samples

oraz odpornooeae na ogieñ oceniano metod¹ TGA, za pomoc¹ kalorymetru sto¿kowego a tak¿e mikrokalorymetru pirolizy i spalania


Véronique Bardinal

R Legros

Chantal Fontaine

Precision Bragg reflectors obtained by molecular beam epitaxy under in situ tunable dynamic reflectometry control

teaching and research institutions in France or abroad, or from public or private research centers.   Precision Bragg reflectors obtained by molecular beam epitaxy under in situ tunable dynamic reflectometry control

synchronizing the material changes and the reflectivity extrema by selecting adequate analysis wavelengths. This technique is shown to provide 885 nm GaAs-AlAs Bragg reflectors with a layer thickness accuracy in excess of 1%.

Distributed Bragg reflectors are key elements in optoelectronic vertical cavity devices like vertical cavity surface emitting lasers 1 and spatial light modulators 2 used in such applications as optical communications, optical interconnects, and optical signal processing. In particular, (Ga,Al)As mirrors are attractive, because GaAs and AlAs are lattice matched and exhibit highly different optical indices.

More-over, non-absorbing Bragg reflectors can be obtained for optical communications wavelengths.

Vertical cavity devices are multilayer structures, which were found to be highly sensitive to the thickness and composition of their unit layers. 1,2 In particular, these errors are detrimental for the Bragg reflectors to be used as elements in vertical cavity devices be-cause the reflector calculation wavelength has to be precisely tuned to the device resonance wavelength within a very tight tolerance. To meet these stringent growth requirements, several in situ thickness monitoring techniques have been developed: reflection high-energy electron diffraction; 3 flux monitoring; 4 spectroscopic ellipsometry; 5 interferometric pyrometry; 6 and dynamic optical reflectometry (DOR). The latter was initially developed with fixed single-wavelength laser light sources: He-Ne lasers (633 nm), 6 laser diodes (670 nm), 7 or argon (514 nm) lasers. 8 More recently, the DOR monitoring accuracy has been improved by the use of broadband sources. 9 In a previous letter, 10 the refractive index dispersion at high temperature measured by DOR with a tunable sapphire-titanium laser used as the light source has been reported by the authors. In this letter, it is shown that this extended DOR technique, called ''tunable dynamic optical reflectometry'' (TDOR), can monitor molecular beam epitaxial (MBE) growth of GaAs-AlAs Bragg reflectors, when associated with an accurate modeling of the reflectivity changes during growth. This technique leads to highly accurate multilayer structures because MBE growth can be monitored under optimized conditions.

The Bragg reflectors designed for this study are centered at 885 nm at room temperature, and consist of a stack of ten periods of GaAs and AlAs alternating layers with quarter-wave thicknesses of 62.3 and 74.3 nm, respectively. These multilayers were grown by MBE in a 3200P Riber system, specially designed for optical in situ monitoring: a viewport is located such that the optical analysis can be performed in the optimal growth geometry which allows high thickness uniformity to be achieved (<1% over 2 in. diameter). Growth details are given elsewhere. 10 A systematic oxide desorption procedure was applied to reproducibly calibrate growth temperature at 600 °C, while Ga/As and Al/As flux ratios were initially optimized by reflection high-energy electron diffraction oscillations. Growth rates of about 1 µm/h were used for both binaries.

For TDOR monitoring, 10 a sapphire-titanium laser beam illuminates via an optical fiber the growing layer surface at nearly normal incidence (2°). Then, after being reflected, it is detected by a Si photodiode.

The signal at the lock-in amplifier output is acquired by a computer also used for laser wavelength tuning via a stepper motor. The wavelength range used for 885 nm Bragg reflector monitoring will be shown to be comprised between 900 and 970 nm. Normal-incidence dynamic reflectance growth monitoring consists of recording the interferences of a light beam reflected from the surface and interfaces of a growing multilayer. As long as growth proceeds, reflectivity evolves with the increase in the uppermost layer thickness. Modeling of reflectivity along growth greatly helps understand the key features of the reflectivity changes. Generally, the optical behavior of a multilayer is modeled through use of the Abele `s matrix formalism, 11 which describes the optical properties of a layer in terms of a characteristic matrix which takes into account layer optical indices and thickness, light wavelength, and incidence angle. A multilayer stack can be represented as an equivalent layer whose matrix is the product of the characteristic matrices of the successive layers in the stack, [START_REF] Macleod | Thin-Film Optical Filters[END_REF][START_REF] Born | Principles of Optics[END_REF] thereby allowing immediate calculation of the reflectance of the multilayer structure. This formalism allows the already grown multilayer fraction to be considered as an equivalent material; thus whatever the materials underneath, they will behave as a single substrate with a single layer growing onto it. During growth of a thick layer, the calculated DOR signal exhibits an oscillator behavior. 10 For transparent materials, all oscillations have the same period equal to /2n. For absorbing materials, the first oscillation is shorter than the following ones, because of interfacial dephasing. The latter is all the more important as the absorbing material is thick. Then substrate absorption is of the utmost importance.

The layers of the 885 nm Bragg reflectors are only slightly thicker than the quarter-wave thicknesses of the standard DOR monitoring wavelengths: 633 (Ref. 6), 670 (Ref. 7), or 514 nm (Ref. 8). In this case, modeling shows the complex oscillatory behavior reflectivity changes along growth, the significant shape being given in Fig. 1(a). Such a variation makes precise control of the growing layer thick-ness, by standard DOR difficult, as the material changes do not correspond to identifiable reflectivity changes. Indeed, with this technique the only possibility of real-time monitoring would lie in the presence of critical points in the oscillation shapes when MBE cell shutters have to be actuated be-cause the expected thickness for the growing material has been reached and the next layer growth has to be initiated. To do this, the reflectivity extrema appear well suited because they can be anticipated by studying the derivative time variation, these critical reflectivity points corresponding to a derivative value of zero. Under these conditions, reflectivity changes along the multilayer growth would exhibit an oscillatory variation, synchronized with the growth of alternating layers, as shown in Fig. 1

(b).

To investigate this promising extended DOR technique, those wavelengths that allow the DOR to be optimized for multilayer growth monitoring were calculated using Abele `s modeling. The geometry of the multilayer to be grown was known at room temperature. Nonetheless, it had to be considered at the growth temperature, 600 °C, used when TDOR would be monitored. Here, the temperature dependence of all the parameters involved in the calculation had to be taken into account. On the one hand, lattice expansion with temperature was calculated to get the layer thicknesses at the growth temperature. On the other hand, dispersion of the GaAs and AlAs optical indices, at that temperature, had to be determined.

Refractive index dispersion for these two binaries had already been determined by TDOR prior to this study in the 750-1000 nm wavelength range of the sapphire:titanium laser. 10 Conversely, the absorption coefficient dispersion which had not yet been characterized was expected to be significant for GaAs, whose gap is shifted to 1.092 µm when temperature rises to 600 °C. [START_REF] Varshni | [END_REF] The absorption index () of a growing layer can be measured from the reflectivity oscillation dampening observed to occur during layer growth. 15 For this study, intermediate AlAs layers were grown prior to GaAs growths in order to get GaAs reflectivity oscillations. 10 As expected, no significant absorption was observed for AlAs.

Dampening of the GaAs reflectivity oscillations was measured for some wavelengths as growth proceeded. The  values derived were 0.125, 0.12, 0.11, and 0.105 for 900, 920, 940, and 960 nm, respectively. These values were com-pared with those obtained by extrapolating Yao's results. 16 Our experimental values were found to be in the same range, whereas the dispersion law appears slightly different.

Following determination of these parameters, the optimal wavelengths to be required for DOR monitoring of the Bragg reflector under study were computed. A wavelength was calculated for each layer, thereby providing a reflectivity extremum for the final layer thickness. The result is shown in Fig. 2. As can be seen, the wavelengths were found to evolve along growth. The wavelengths required for TDOR monitoring of 885 nm Bragg reflectors ranges from 900 to 970 nm, and, as expected, are well suited to the use of a sapphire-titanium laser. Their sharp initial decrease originates from the GaAs substrate absorption which causes a significant interfacial dephasing. As soon as some alternating layers are grown, the substrate effect decreases and wavelengths quickly tend to an asymptotic value. The latter corresponds to that of the reflector which progressively is formed during growth as evidenced by the progressive increase in the reflectivity maxima towards 1 in Fig. 1(b). This multilayer, an exact Bragg reflector at room temperature, is now an asymmetrical reflector at 600 °C. 17 Indeed, layer thicknesses are no longer /4n as a result of the difference in GaAs and AlAs refractive index temperature dependence.

These optimal TDOR monitoring wavelengths were used to grow 885 nm GaAs-AlAs reflectors.

Growth was performed under TDOR monitoring; at the beginning, the laser was initially tuned to the initial wavelength calculated for monitoring the first layer thickness. Afterwards, the cell shutters were alternatively opened and closed every time the reflectivity derivative was observed to reach 0.

Simultaneously, the laser was tuned to the next calculated wavelength in order to provide the optimal monitoring of the new layer being grown. The reflectivity spectrum of the grown Bragg reflector at room temperature is given in Fig. 3. The calculated spectrum is also plotted for comparison purposes. As expected, a good agreement is obtained between both spectra, indicating a thickness precision in excess of 1%.

To conclude, we have presented a new dynamic optical reflectometry technique based on the use of a tunable laser which allows real-time monitoring of layer thicknesses grown by MBE. It has resulted in a precise monitoring (<1%) of thicknesses by synchronizing the change in material with reflectivity extrema. This technique should be of great interest for monitoring the growth of all multilayer structures used in optoelectronics devices where stringent requirements are imposed on multilayer geometry.

Finally, it should be noted that TDOR paves the way for complete MBE growth automation, as it makes it possible to drive cell shutters changes from reflectivity analysis.

FIG. 1 .

 1 FIG. 1. Reflectivity variation during 600 °C growth of a Bragg reflector centered at 885 nm at room temperature: (a) fixed monitoring wavelength of 633 nm. (b) ideal monitoring conditions. (c) Reflectivity derivative variation for case (b).

FIG. 2 .

 2 FIG.2. Optimal wavelengths to be used for monitoring the successive layer thicknesses.

Acknowledgements

The authors gratefully acknowledge G. Lacoste for technical assistance and A. Mun ˜oz-Yagu ¨e for fruitful discussions.