Marielle Margier

Tiffany Antoine

Aurélie Siriaco

Marion Nowicki

Charlotte Halimi

Matthieu Maillot

Stéphane Georgé

Emmanuelle Reboul
email: emmanuelle.reboul@univ-amu.fr.

The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability

Keywords: legumes, carotenoids, fibers, phytates, saponins, tannins, bioaccessibility, intestinal absorption

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

This article is protected by copyright. All rights reserved.

The presence of prepared pulses within a meal could alter both the bioaccessibility and the uptake of fat-soluble vitamins. This effect was dependent on pulse type and/or on pulse cooking method, likely because pulse contents in molecules with lipid-lowering properties such as fibers, phytates, saponins and tannins were different between these conditions. Experiments with purified fibers, phytates, saponins and tannins strongly suggested that these compounds were responsible for the decrease of fat-soluble vitamin bioavailability.

Our results thus underline the importance of consuming pulses within micronutrient-rich meals/ recipes.

Introduction

Fat-soluble vitamins (i.e. vitamin A, D, E and K) are essential molecules playing key roles in the development and the perenniality of our organism. The occurrence of fat-soluble vitamin deficiencies is a major public health issue in developing countries, as these deficiencies can lead to blindness and infections [START_REF] Wiseman | The vicious cycle of vitamin a deficiency: A review[END_REF], rickets [START_REF] Arabi | Hypovitaminosis D in developing countries-prevalence, risk factors and outcomes[END_REF], neurologic defects [START_REF] Dror | Vitamin E deficiency in developing countries[END_REF], as well as hemorrhagic diseases of the newborn [START_REF] Mcninch | Vitamin K deficiency bleeding: early history and recent trends in the United Kingdom[END_REF]. Fat-soluble vitamin (principally vitamin D and E) sub-deficiencies and/ or insufficient intakes have also been described in Western countries [START_REF] Hilger | Micronutrient Intake in Healthy Toddlers: A Multinational Perspective[END_REF][START_REF] Vinas | Projected prevalence of inadequate nutrient intakes in Europe[END_REF]. The correlation between these subdeficiencies and the growing incidence of chronic pathologies including cardiovascular, neurodegenerative and bone pathologies is presently widely explored because several studies reported a causative association [START_REF] Holick | The vitamin D deficiency pandemic: Approaches for diagnosis, treatment and prevention[END_REF][START_REF] Azzi | The rise, the fall and the renaissance of vitamin E[END_REF].

Extensive investigation over the past decades focused on the factors regulating fat-soluble vitamin absorption. Indeed, even if these vitamins are brought in adequate amounts in the diet, their health effects depend on their bioavailability, which can be modulated by interactions with other meal components. For example, it has been showed that fat could act as an effector of fat-soluble micronutrient absorption, while polyphenols, phytosterols or fibers could alter their bioavailability (reviewed in [START_REF] Reboul | Absorption of vitamin A and carotenoids by the enterocyte: focus on transport proteins[END_REF][START_REF] Reboul | Intestinal absorption of vitamin D: from the meal to the enterocyte[END_REF][START_REF] Reboul | Vitamin E Bioavailability: Mechanisms of Intestinal Absorption in the Spotlight[END_REF]).

Besides, for the growing human population in a world with limited resources, one vital challenge today is the ability to make diets sustainable for all. Pulses may constitute a part of the solution.

Indeed, additionally to their environmental benefits [START_REF] Gan | Diversifying crop rotations with pulses enhances system productivity[END_REF], they are a good source of proteins, fibers, micronutrients and bioactive phytochemicals [START_REF] Bouchenak | Nutritional quality of legumes, and their role in cardiometabolic risk prevention: a review[END_REF]. It has also been shown that regular consumption of pulses can lead to decreased plasma cholesterol and triglyceride levels [START_REF] Anderson | Pulses and lipaemia, short-and long-term effect: Potential in the prevention of cardiovascular disease[END_REF], suggesting an impact on lipid absorption and/or metabolism. This impact is likely due to pulse fiber content [START_REF] Asif | Application and opportunities of pulses in food system: a review[END_REF], but other pulse bioactive compounds such as phytates, saponins or polyphenols may also play important roles in this phenomenon [START_REF] Bouchenak | Nutritional quality of legumes, and their role in cardiometabolic risk prevention: a review[END_REF]. There is little clear data on the effects of phytates on lipid digestionabsorption process. However, a recent study in chickens showed that phytates could affect lipase Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

activity and lipid metabolism [START_REF] Liu | Effect of dietary sodium phytate and microbial phytase on the lipase activity and lipid metabolism of broiler chickens[END_REF]. In addition, saponins have often been described as anti-obesity compounds [START_REF] Han | Saponins from platycodi radix ameliorate high fat diet-induced obesity in mice[END_REF], notably by inhibiting the activity of pancreatic lipase [START_REF] Zhao | Antiobese and hypolipidemic effects of platycodin saponins in diet-induced obese rats: evidences for lipase inhibition and calorie intake restriction[END_REF]. Saponins may also limit the micellar incorporation of cholesterol [START_REF] Chavez-Santoscoy | Effect of flavonoids and saponins extracted from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors[END_REF]. The lipid-lowering properties of pulses constitute another reason to promote their consumption. However, these data suggest that pulses can also have an impact on fat-soluble vitamin absorption. Interestingly, the cooking method can modulate the bioactive compound content of pulses [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF]. Pulse cooking method may consequently modulate the putative effect of pulses on fat-soluble vitamin absorption.

The aim of our study was thus to i) assess the impact of pulses (kidney beans, white beans, chickpeas, green/ brown lentils and flageolets) and their preparation modes (home cooking vs.

canning) on the bioavailability of vitamin A (retinyl palmitate and its precursor: -carotene), D (cholecalciferol), E (-tocopherol) and K (phylloquinone); and ii) to decipher the molecular mechanisms underlying these effects.

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

Materials and methods

Chemicals

Retinyl palmitate, cholecalciferol, -tocopherol, phylloquinone and β-carotene (all > 95% pure), as well as 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (phosphatidylcholine), 1-palmitoyl-snglycero-3-phosphocholine (lysophosphatidylcholine), monoolein, free cholesterol, oleic acid, sodium taurocholate, fibers (cellulose), phytates (phytic acid sodium salt hydrate), saponins (purified quillaia saponins) and tannins (grape seeds oligomeric proanthocyanidins) were purchased from Sigma Aldrich. Refined olive oil was generously provided by Dr Marie-Josèphe Amiot (UMR Moisa, Montpellier, France). Minced beef (with 5% of fat) and potatoes were purchased from a local supermarket (Casino, France). Kidney beans (Phaseolus Vulgaris L.), white beans (Phaseolus Vulgaris L.), chickpeas (Cicer Arietinum), green lentils (Lens culinaris M.), brown lentils (Lens culinaris M.) and flageolets (Phaseolus vulgaris L.) were purchased at CIACAM society (Vitrolles, France). Note that the pulses used in this study were from the same lots than the pulses fully characterized previously [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF].

Pulse preparation

Pulses were either cooked following a household method (Table 1) or canned (Table 2) according to standard protocols [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF]. To respect traditional eating habits, green lentils and flageolets were prepared following the household-cooking method while brown lentils were canned. Kidney beans, white beans and chickpeas were prepared with both methods, allowing to compare the effect of the cooking method on fat-soluble vitamin bioavailability.

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

Fiber, phytate, saponin and tannin measurements in pulses and pureed potatoes

Fibers, phytates, saponins and tannins had previously been assayed in the different prepared pulses [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF]. Fibers, phytates, saponins and tannins from potatoes were assayed following the same methods.

In vitro digestion experiment

The meals used in the first set of in vitro digestion experiments were made of 1.2 g of pan-cooked minced beef meat, 200 L of refined olive oil and either 6.7 g of pureed potatoes (control) or 6.7g of cooked pulses. The meals used in the second set of experiments were made of 1.2g of minced beef meat, 200 L of refined olive oil and 6.7 g of potatoes, and were supplemented with purified cellulose (0.7mg), phytates (42.5 mg), saponins (6 mg) or tannins (0.4 mg). These amounts were specifically chosen to mimic on average those brought in the test meals by prepared pulses ( [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF],

Table 3). Beef and refined olive oil brought minor amounts of fat-soluble vitamins to the meal, which has no consequence on the interpretation of the data as these two ingredients were present in all conditions. Each test meal was supplemented with 200 ng of fat-soluble vitamins dissolved in the refined olive oil. The in vitro digestion experiment was conducted as previously described [START_REF] Malapert | beta-Cyclodextrin Does not Alter the Bioaccessibility and the Uptake by Caco-2 Cells of Olive By-Product Phenolic Compounds[END_REF][START_REF] Reboul | Bioaccessibility of carotenoids and vitamin E from their main dietary sources[END_REF].

The final mixture representing the digesta was centrifuged (2500 g, 1 h, 10 °C) to separate the aqueous fraction containing the mixed micelles. Aliquots of digestas and mixed micelles were taken and stored at -80°C under a nitrogen atmosphere until analysis. Bioaccessibility was calculated as the percentage of vitamin recovered in mixed micelles divided by the amount of vitamin recovered in the digesta at the end of the digestion. Note that micellar vitamin A, initially provided in the form of retinyl palmitate, was mainly recovered as retinol.

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

Fat-soluble vitamin absorption by intestinal cells in culture

Preparation of micelles from in vitro digestion

According to preliminary cytotoxicity tests, fat-soluble vitamin micelles obtained from in vitro digestions were diluted in DMEM (1:7; vol:vol).

Preparation of synthetic mixed micelles enriched in vitamin K

Synthetic mixed micelles were prepared as previously described [START_REF] Reboul | Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI)[END_REF]. Briefly, appropriate volumes of phosphatidylcholine, lysophosphatidylcholine, monoolein, cholesterol, oleic acid and vitamin K stock solutions were transferred to glass bottles to obtain the following final concentrations: 0.04 mM, 0.16 mM, 0.3 mM, 0.1 mM, 0.5 mM and 5 µM, respectively. Stock solution solvents were carefully evaporated under nitrogen. Dried residue was solubilized in DMEM containing 5 mM taurocholate and vigorously mixed by 25 watt sonication (Branson 250W sonifier, Osi) for 3 min. The mixtures obtained were sterilized and filtered by passing them through a presterilized 0.22 µm filter. The concentration of vitamin K in the micellar solutions was measured before each experiment. The day of the experiments, mixed micelles were supplemented or not in cellulose, phytates, saponins and/or tannins, at the following concentrations: 212 µg/mL, 30 µg/mL, 2 µg/mL and 3.5mg/mL, respectively, to be in accordance with the mean levels putatively recovered in micelles after pulse digestion (Table 3 [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF]).

Cell experiments

Caco-2 clone TC7 cells were cultured as previously described [START_REF] Goncalves | Pinoresinol of olive oil decreases vitamin D intestinal absorption[END_REF]. Twelves houses prior each This article is protected by copyright. All rights reserved.

fat-soluble vitamins, scraped and collected in 0.5 mL ice-cold PBS. Absorbed vitamin was calculated as the percentage of the amount of vitamin recovered in cells divided by the total amount of vitamin delivered to the cells (i.e. vitamin found in the medium plus vitamin remaining in the cells at the end of the experiment). Note that due to the short incubation time (1h), vitamin A was recovered in cells as retinol only. All the samples were stored at -80°C until analysis.

Fat-soluble vitamin extraction

The internal standards were retinyl acetate for samples containing retinyl palmitate and vitamin D 3 , echinenone for samples containing vitamin K, tocopheryl nicotinate for samples containing vitamin E and apo-8'-carotenal for samples containing -carotene, respectively. Samples were extracted as previously described [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters[END_REF]. The dried extract was dissolved in 200 µL of methanol-dichloromethane (65:35; vol:vol). A final volume of 50 µL for micelle samples, 20 µL for digesta samples, 180 µL for the cellular sample were used for HPLC analysis. The fat-soluble vitamins were separated as previously described [START_REF] Gleize | Simple and fast HPLC method for simultaneous determination of retinol, tocopherols, coenzyme Q(10) and carotenoids in complex samples[END_REF]. All molecules were identified by retention time compared with standards by UVvisible detection.

Statistical analysis

For all groups, data were expressed as mean ± SEM. Statistical analyses were performed using SPSS (SPSS Inc., Chicago, USA). Prior to ANOVA (fixed-effects models), data were tested for equality of variances. The distribution of all dependent variables was graphically checked and was considered as This article is protected by copyright. All rights reserved.

Results

Potatoe and pulse composition in fibers, phytates, saponins and tannins.

We previously showed that 100 g of drained cooked pulses contained 3.8 g to 13.8 g of dietary fibers, 386 to 714 mg of phytates, 99 mg to 175 mg of saponins and 1.8 to 16.6 mg of tannins, depending on both pulse type and pulse cooking method [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF]. In comparison, we showed that 100g of cooked potatoes contained 2.2  0.0 g of dietary fibers, 141.5  19.6 mg of phytates, 32.3  1.3 mg of saponins, and no tannins. These results are summarized in Table 3.

Bioaccessibility of fat-soluble vitamins is decreased in the presence of legumes and this

effect is modulated by the cooking method.

The bioaccessibility of the different fat-soluble vitamins were significantly different from each other within the control meal: vitamin E (≈ 41%) > vitamin D (≈ 37%) > vitamin K (≈ 28%) > retinyl palmitate (≈ 24%) > β-carotene (≈ 12%) (Figure 1).

In most of the experiments, the bioaccessibility of fat-soluble vitamins was impaired in the presence of household pulses compared to the control meal. This decreased was dependent on pulse types and on fat-soluble vitamins. For instance, the decrease in retinyl palmitate bioaccessibility ranged from 43% in the presence of flageolets (p=0.042) to 64% in the presence of white beans (p=0.001).

The bioaccessibility of β-carotene was decreased from 42% in presence of white beans (p=0.122) to 65% in presence of kidney beans (p=0.004). The bioaccessibility of vitamin D was decreased by 34% and 45% in the presence of chickpeas (p=0.03) and lentils (p=0.002), respectively. Similarly, the bioaccessibility of vitamin E was significantly decreased from 45% in presence of kidney beans (p=0.005) to 50% in presence of flageolets (p=0.001). Finally, the decrease in vitamin K

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

bioaccessibility was decreased from 47% in presence of flageolets (p=0.015) to 67% in presence of chickpeas (p=0.001).

Interestingly, in some experiments, results showed that canned pulses were less deleterious than household cooked pulses. For example, the bioaccessibility of β-carotene was higher in the presence of canned chickpeas compared to household-cooked chickpeas (+111%, p=0.023). The bioaccessibility of retiol/ retinyl palmitate was also more important in presence of canned white beans than in the presence of household-cooked white beans (+68.4%, p=0.05). Finally, vitamin K bioaccessibility was also increased by 78.4% in the presence of canned white beans compared to household-cooked white beans (p=0.05).

Fat-soluble vitamin uptake by Caco-2 cells is decreased in the presence of micelles from in vitro digestion of test meals containing pulses compared to control micelles.

The micelles obtained from the in vitro digestion of the test meals containing either potatoes or prepared pulses were then incubated on Caco-2 cells monolayers after dilution in cell culture media.

The uptake of the different fat-soluble vitamins after 1h of incubation was significantly different from each other within the control meal: vitamin A (≈15%) > vitamin E (≈11%) > vitamin D (≈10%) > vitamin K (≈4%) > β-carotene (≈1%) (Figure 2).

The presence of micelles from in vitro digestion of meals containing pulses decreased the uptake of fat-soluble vitamins, but not that of -carotene, compared to control micelles (p<0.05). The uptake of preformed vitamin A was significantly decreased by -39.8% in the presence of micelles isolated from household-cooked white bean in vitro digestion (p=0.018). The uptake of vitamin D was decreased from -26.3% in the presence of micelles from digested meals containing flageolets (p=0.012) to -66.7% in the presence of micelles from digested meals containing chickpeas (p=0.01).

The uptake of vitamin E was significantly decreased by -34.4% in the presence of micelles isolated This article is protected by copyright. All rights reserved.

from canned kidney bean in vitro digestion (p=0.001). The uptake of vitamin K was also decreased by -47.3% to -56.7% in the presence of micelles from digested meals containing kidney beans (p=0.034)

or lentils (p=0.007) compared to the control micelles.

Conversely to what was observed for bioaccessibility, pulse cooking method had no clear effect on fat-soluble vitamins uptake.

Fat-soluble vitamin bioaccessibility and uptake are negatively correlated to the presence of fibers, phytates, saponins or tannins

The first and the second principal components of the PCA drove 41.3% and 15.5% of the total variance, respectively. Based on the first axe, PCA showed that the presence of phytates, saponins, tannins and fibers were inversely correlated to the bioaccessibility and uptake of all vitamins, and especially both the bioaccessibility and the uptake of vitamin D, E and K, as well as the bioaccessibility of vitamin A (retinyl palmitate) (Figure 3A). On the first axe, potatoes (low in fibers, phytates, saponins and tannins) were opposed to all pulses (Figure 3B). Based on the second axe, PCA showed that the presence of fibers was inversely correlated to -carotene bioaccessiblility and uptake, and to retinol uptake; and that the presence of tannins was inversely linked to vitamin D bioaccessibility and uptake and to vitamin K bioaccessiblity.

We thus performed further experiments using purified fibers, phytates, saponins and tannins to confirm their effects of fat-soluble vitamin bioavailability.

Vitamin K bioaccessibility is decreased in the presence of fibers, tannins and saponins.

We specifically assessed the impact of fibers, phytates, saponins and tannins on the bioaccessibility of vitamin K (used as a model vitamin).

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

As shown in Figure 4, the addition of pulse compounds (saponins, tannins and fibers) in a control meal significantly decreased the bioaccessibility of vitamin K. Saponins were the most deleterious, with a reduction in vitamin K bioaccessibility of 60.9% (p<0.0001). This reduction was less important in presence of tannins and fibers (-34.4%, p=0.0002 and -13.4%, p=0.037 respectively). Finally, the bioaccessibility of vitamin K was not decreased in the presence of phytates.

Vitamin K uptake by Caco-2 cells is decreased in the presence of fibers, phytates, saponins and tannins, but these effects are not additive.

The impact of fibers, phytates, saponins and tannins, alone or in combination, on vitamin K uptake (used as a model vitamin) by Caco-2 cells was finally evaluated (Figure 5).

 Impact of one compound (fibers or phytates or saponins or tannins)

Vitamin K uptake was significantly decreased when Caco-2 cells were incubated with synthetic mixed micelles containing fibers or phytates or saponins. Our results showed that saponins had the most deleterious effect: vitamin K uptake was decreased by 22.5% compared to the control condition (p<0.001). The presence of fibers and phytates in synthetic mixed micelles also decreased vitamin K uptake by 14.8% (p=0.01) and 11.9% (p=0.05), respectively. Tannins alone had no effect on vitamin K uptake compared to the control condition.

 Impact of two compounds

The presence of two pulse compounds in synthetic mixed micelles was less deleterious than the presence of a single compound. This decrease varies from 2.1% to 18.0% depending on the conditions. Tannin + saponins decreased vitamin K uptake by 18.0% compared with the control condition (p=0.0005). Similarly, fibers + saponins decreased vitamin K uptake by 14.5% (p=0.013). In the presence of tannins, fibers did not impact vitamin K uptake anymore. Similarly, the addition of This article is protected by copyright. All rights reserved.

phytates to another compound (saponins or tannins or fibers) in the synthetic mixed micelles did not induced a reduction of vitamin K uptake compared to the control condition.

 Impact of three compounds

The presence of saponins in a combination of 3 pulse compounds appeared to reduce vitamin K uptake. Indeed, saponins + tannins + phytates and saponins + tannins + fibers decreased vitamin K uptake by 18.2% (p=0.0009) and 15.3% (p=0.0063), respectively, compared to the control.

Conversely, phytates + fibers + tannins and phytates + fibers + saponins conditions had no effect on vitamin K uptake.

 Impact of four compounds:

Finally, the combination of 4 pulses compounds (fibers, phytates, saponins and tannins) significantly decreased vitamin K uptake (-14.5%; p=0.013).

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

Discussion

Pulses components are acknowledged to impair mineral bioavailability [START_REF] Sandberg | Bioavailability of minerals in legumes[END_REF], as well as to reduce lipid absorption [START_REF] Anderson | Pulses and lipaemia, short-and long-term effect: Potential in the prevention of cardiovascular disease[END_REF]. However, no data are available on the effect of pulses on fat-soluble vitamin and carotenoid digestion-absorption. This study thus aimed at assessing and deciphering the effect of pulses on the 2 main steps governing fat-soluble vitamin bioavailability, i.e. their transfer to mixed micelles (bioaccessibility) and their entry into the intestinal cell (uptake by the enterocyte).

Bioaccessibility was evaluated using an in vitro digestion model, while uptake by enterocyte was evaluated using Caco-2 cells in culture.

In vitro digestions were first performed with test meals containing meat and refined olive oil, and completed with either potatoes or pulses as a side dish. In most cases, the presence of pulses (kidney beans, white beans, chickpeas, green or brown lentils, flageolets) within a test meal reduced fat-soluble vitamin and carotenoid bioaccessibility (up to -65% for β-carotene, -69% for retinylpalmitate, -45% for vitamin D, -53% for vitamin E and -67% for vitamin K) compared to a test meal containing potatoes. Different pulse components can be responsible for this effect. PCA analysis showed that fibers, phytates, saponins and tannins from pulses were associated to this negative effect for most of the vitamins. Interestingly, in some case, this negative effect was less important when canned pulses were used compared to paired household-cooked pulses. This can be related to the fact that canned pulses presented a different composition than household-cooked pulses -and in particular less fibers, less phytic acid and sometimes less tannins, due to an increased leaching of water-soluble compounds into the broth during canning process [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF].

To further explore the specific effect of each compound, we then conducted in vitro digestions with test meals containing potatoes and supplemented with either purified cellulose, phytates, saponins or tannins, in amounts representing those measured on average in cooked pulses [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF].

These in vitro Molecular Nutrition & Food Research

This article is protected by copyright. All rights reserved.

digestions were performed with vitamin K as a model vitamin, as vitamin K behavior was shown to statistically represent most of the other vitamins (data not shown).

Cellulose was chosen as a fiber model because it is a major fiber in pulses, representing about one half of total dietary fiber in different legume species [START_REF] Marlett | Content and composition of dietary fiber in 117 frequently consumed foods[END_REF]. It has previously been shown that dietary fibers could bind micellar component such as bile salts, phospholipids or cholesterol in vitro [START_REF] Vahouny | Dietary fibers: V. Binding of bile salts, phospholipids and cholesterol from mixed micelles by bile acid sequestrants and dietary fibers[END_REF],

and that fibers could impact on the absorption of some carotenoids but not that of vitamin E [START_REF] Riedl | Some dietary fibers reduce the absorption of carotenoids in women[END_REF]. In our study, we found a significant but modest effect of cellulose on vitamin K bioaccessibility. This confirms that fiber effect on fat-soluble vitamin bioaccessibility is likely moderate.

The second pulse component that can impact on fat-soluble bioaccessibility is phytic acid, due to its potential ability to inhibit lipase activity in the pancreas or small intestine of broilers fed with a high phytate diet [START_REF] Liu | Effect of dietary sodium phytate and microbial phytase on the lipase activity and lipid metabolism of broiler chickens[END_REF]. However, when added to a meal containing meat and potatoes, phytates did not reduce vitamin K bioaccessibility. This lack of effect may be due to potential interaction of phytates with meal proteins during the gastric digestion step [START_REF] Kies | Interaction between protein, phytate, and microbial phytase. In vitro studies[END_REF], which would limit phytate interactions with lipases.

The third compound that could explain this effect is saponins. Indeed, saponins were shown to inhibit the in vitro activity of the pancreatic lipase, and a diet enriched in 35 to 75 mg of saponins / kg increased triglyceride outputs in rat feces [START_REF] Zhao | Antiobese and hypolipidemic effects of platycodin saponins in diet-induced obese rats: evidences for lipase inhibition and calorie intake restriction[END_REF]. Our data showed that saponins were the compounds with the higher inhibitory effect on vitamin K bioacessibility. We suggest that this effect is linked to their ability to limit the incorporation of lipids such as cholesterol into mixed micelles [START_REF] Chavez-Santoscoy | Effect of flavonoids and saponins extracted from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors[END_REF].

Finally, the last compounds that could explain the inhibitory effect of pulses are tannins. The addition of tannins at fairly low concentrations in the test meal was sufficient to shown a significant reduction of vitamin K bioaccessibility. This inhibitory effect is likely due to an interaction of tannins with pancreatic lipase [START_REF] Gu | Inhibition of key digestive enzymes by cocoa extracts and procyanidins[END_REF][START_REF] Oliveira | Inhibition of Pancreatic Lipase and Triacylglycerol Intestinal Absorption by a Pinhao Coat (Araucaria angustifolia) Extract Rich in Condensed Tannin[END_REF].

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

We then explored the effect of pulses on fat-soluble vitamin uptake by differentiated Caco-2 cell monolayers. This popular enterocyte-like cell model has previously been used widely to investigate fat-soluble vitamin absorption [START_REF] Desmarchelier | Comparison of the Micellar Incorporation and the Intestinal Cell Uptake of Cholecalciferol, 25-Hydroxycholecalciferol and 1-alpha-Hydroxycholecalciferol[END_REF][START_REF] Goncalves | Intestinal scavenger receptors are involved in vitamin K1 absorption[END_REF][START_REF] Borel | This article is protected by copyright[END_REF][START_REF] Reboul | ATP-binding cassette transporter A1 is significantly involved in the intestinal absorption of alpha-and gammatocopherol but not in that of retinyl palmitate in mice[END_REF]. The presence of pulse components in micelles isolated from in vitro digestions altered fat-soluble vitamin uptake (-40% for retinyl-palmitate, -67% for vitamin D, -50% for vitamin E and -57% for vitamin K). This effect was independent of the cooking method.

We showed in a previous work that micellar fatty acids could impact on vitamin D absorption by modifying mixed micelle physicochemical properties. In particular, fatty acid micellar composition could modulate the size and the electric charge of mixed micelles [START_REF] Goncalves | Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells[END_REF], which in turn impacted on micelle ability to bind the extracellular loop of membrane transporters involved in vitamin D intestinal uptake [START_REF] Goncalves | Micellar lipid composition affects micelle interaction with class B scavenger receptor extracellular loops[END_REF]. In this study, the presence of potatoes vs different cooked pulses in the test meal did not induce any change in the size and electric charge of the micelles obtained from in vitro digestions (data not shown). The observed inhibitory effect on fat-soluble vitamin uptake was thus likely due to the presence of specific pulse compounds in either the extramicellar or the micellar phase.

PCA analysis highlighted that fat-soluble vitamin uptake was also negatively associated with fibers, phytates, saponins and tannins. To confirm these associations and specifically address the effect of each compound on fat-soluble vitamin uptake process by the enterocytes, we assessed the uptake by Caco-2 cells of micellar vitamin K (used a s a model vitamin) in the absence or presence of these purified pulse compounds. We used vitamin K-rich synthetic mixed micelles supplemented with pulse compounds and not micelles from in vitro digestion to better control purified compound concentrations. The compounds were added alone or in combination in concentrations mimicking those theoretically found in the micellar phase after pulse digestion. Our results showed a deleterious effect of those compounds on fat-soluble vitamin uptake as follows: saponins > fibers > phytates.

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul This article is protected by copyright. All rights reserved.

The strong effect of saponins on vitamin K uptake is not surprising. Indeed, a diet enriched in saponins (0.9%) induced an increased cholesterol excretion in chicks, as well as a defect in vitamin A and E absorption [START_REF] Jenkins | Effects of dietary saponins on fecal bile acids and neutral sterols, and availability of vitamins A and E in the chick[END_REF]. Moreover saponins were shown to inhibit carrier-mediated nutrient transport in enterocytes [START_REF] Johnson | Influence of saponins on gut permeability and active nutrient transport in vitro[END_REF]. Saponins may thus interact with fat-soluble vitamin transporters such as the Scavenger Receptor Class B type I (SR-BI) or the NPC1-like 1 transporter (NPC1L1), that are ubiquitous fat-soluble micronutrient transporters [START_REF] Reboul | Vitamin E Bioavailability: Mechanisms of Intestinal Absorption in the Spotlight[END_REF]. The effect of fibers is likely due to their ability to bind micellar lipids [START_REF] Vahouny | Dietary fibers: V. Binding of bile salts, phospholipids and cholesterol from mixed micelles by bile acid sequestrants and dietary fibers[END_REF], as mentioned above. The effect of phytates at the enterocyte levels was unexpected and require further investigations to identify the underlying mechanisms. Finally, tannins had no effects on vitamin K absorption. We showed in previous studies that polyphenols could inhibit fat-soluble vitamin and carotenoid bioavailability. In particular, naringenin was shown to inhibit vitamin E [START_REF] Reboul | Effect of the main dietary antioxidants (carotenoids, gamma-tocopherol, polyphenols, and vitamin C) on alpha-tocopherol absorption[END_REF] and lutein [START_REF] Reboul | Differential effect of dietary antioxidant classes (carotenoids, polyphenols, vitamins C and E) on lutein absorption[END_REF] absorption. Additionally, olive oil polyphenols, and especially pinoresinol, also decreased vitamin D intestinal absorption in rats and Caco-2 cells [START_REF] Goncalves | Pinoresinol of olive oil decreases vitamin D intestinal absorption[END_REF]. The lack of effect of tannins in this study may be due to the low concentration used to mimic tannin concentration in pulses. It can also be due to a lack of interactions of tannins with membrane transporters. Indeed, these proteins are usually highly glycosylated [44, 45], which may partly protect them against a complexation with tannins.

Interestingly, the effects of the different compounds were not additive. For instance, the presence of fibers + phytate or fibers + tannins, showed no effect on vitamin K uptake, while fibers or phytates alone were deleterious. This highlight the fact that the compounds can probably interact with each other, which has already been described for fibers and polyphenols [46]. Finally, it worth to mention that the presence of saponins induces a significant decrease in vitamin K uptake in 6/7 conditions, showing that saponins are likely very potent inhibitors of fat-soluble micronutrient uptake.

After being taken up by the enterocytes, fat-soluble vitamins are incorporated into chylomicrons to be released to the lymph. Further investigations are needed to determine whether pulse components can modulate this last step of fat-soluble vitamin absorption process as well. This article is protected by copyright. All rights reserved.

Overall, our results show for the first time that the presence of pulses within a meal can decrease fat-soluble vitamin and carotenoid bioavailability, by impairing both their bioaccessibility and their uptake by intestinal cells. These effects are likely due to the presence of compounds responsible for the lipid-lowering effect of pulses, i.e. fibers, phytates, saponins and tannins. Further investigations are needed to i) verify that the specific effects of these compounds observed on vitamin K bioavailability can be extended to other fat-soluble vitamins, ii) fully understand the molecular mechanisms underlying such effects, and iii) identify the interactions modulating the effects of these compounds, including when pulses are cooked together with meat and oil and not just added as a side dish afterwards. Moreover, as pulse cooking method can modulate their fiber and phytate contents [START_REF] Margier | Nutritional Composition and Bioactive Content of Legumes: Characterization of Pulses Frequently Consumed in France and Effect of the Cooking Method[END_REF], which can in turn have an impact on fat-soluble vitamin bioavailability, additional research is required to set up household or industrial preparation processes limiting the presence of unwanted compounds without altering pulse nutritional quality. Finally, our results should be taken This article is protected by copyright. All rights reserved.

[44] Miura, S., Saku, K., Ezetimibe, a selective inhibitor of the transport of cholesterol. Intern Med 2008, 47, 1165-1170.

[45] Shen, W. J., Hu, J., Hu, Z., Kraemer, F. B., Azhar, S., Scavenger receptor class B type I (SR-BI): a versatile receptor with multiple functions and actions. Metabolism 2014, 63, 875-886. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Tables

Table 3 Data are means ± SD (n = 3-4). An asterisk indicates a significant difference (p < 0.05) between the two cooking methods . n.d. = not detectable or below the limit of quantitation in the sample. This article is protected by copyright. All rights reserved. At the beginning of the experiment, the apical side received synthetic mixed micelles enriched with 5 µM of vitamin K and supplemented or not with specific pulse compounds. Phytates, saponins, tannins and cellulose were added at the following concentrations: 212 µg/mL, 30 µg/mL, 2 µg/mL and 3.5mg/mL, respectively. Cells were then incubated 1 h at 37°C. Absorbed vitamin K was calculated as the percentage of the amount of vitamin recovered in cells divided by the total amount of vitamin delivered to the cells (i.e vitamin found in the medium plus vitamin remaining in the cells at the end of the experiment). Data are means ± SEM (n=3 per condition). * p<0.05, ** p<0.01, ** p<0.001 when compared with control (without specific pulse compounds).

  normal. Tukey's test was used as a post-hoc test for pairwise comparisons. A principal Component Analysis (PCA) was conducted on all samples (including replicates) and all variables to analyze the relationship between the presence of fibers/ phytates/ saponins/ tannins and fat-soluble vitamin bioaccessibility and uptake.Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S.,Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 www.mnf-journal.com Page 10 Molecular Nutrition & Food Research

  Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S.,Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 www.mnf-journal.com Page 14 Molecular Nutrition & Food Research

  , E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323

  Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323

  into account by food companies to propose pulse-based products displaying a sufficient micronutrient-density, as well as by Public Health Agency to promote pulse consumption in a wellbalanced diet rich in micronutrients. Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 www.mnf-journal.com Page 20 Molecular Nutrition & Food Research This article is protected by copyright. All rights reserved. Acknowledgement Authors declare no conflict of interest. Authors are very grateful to Ndèye Y. Ba (C2VN, Marseille) for valuable technical help. Marielle Margier received a doctoral fellowship (2015-2018) from Region PACA in partnership with the Centre Technique de Conservation des Produits Alimentaires (CTCPA Avignon). This work was partly supported by an INRA Priority Action of the "Alimentation Humaine" department (2018-2020). The project leading to this publication has also received funding from Excellence Initiative of Aix-Marseille University -A*MIDEX, a French "Investissements d'Avenir" program. Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323

[ 46 ]

 46 Bohn, T., Dietary factors affecting polyphenol bioavailability. Nutr Rev 2014, 72, 429-452. Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323 www.mnf-journal.com Page 27 Molecular Nutrition & Food Research

Figure 1 :

 1 Figure 1: Effect of pulses on fat-soluble vitamin bioaccessibility vitro experiments were performed with test meals containing minced beef meat, refined olive oil supplemented with fat-soluble vitamins, and either potatoes (control condition, white bar), household cooked pulses (black bar) or canned pulses (striped bar). Vitamin bioaccessibility was calculated as the percentage of vitamin recovered in mixed micelles divided by the amount of vitamin recovered in the digesta at the end of the digestion. Data are means ± SEM (n=4 per condition). Means values followed by a different superscript letter are significantly different (p< 0.05).

Figure 2 :

 2 Figure 2: Effect of pulses on fat-soluble vitamin uptake by Caco-2 cellsAt the beginning of the experiment, the apical side received fat-soluble vitamin-rich micelles obtained from in vitro digestions of meal containing either potatoes (white bar), household cooked pulses (black bar) or canned pulses (striped bar) at a 1:7 dilution. Cells were then incubated 1 h at 37°C. Absorbed vitamin was calculated as the percentage of the amount of vitamin recovered in cells divided by the total amount of vitamin delivered to the cells (i.e vitamin found in the medium plus vitamin remaining in the cells at the end of the experiment). Data are means ± SEM (n=4 per condition). Means values followed by a different superscript letter are significantly different (p< 0.05).

Figure 3 :

 3 Figure 3 : Relashionship between fat-soluble vitamin bioaccessiblity/ uptake and meal content in phytates, saponins ans tannins.A principal Component Analysis (PCA) was conducted on all samples (potatoes (x), householdcooked kidney beans (○), canned kidney beans (•), household cooked white beans (□), canned white beans (■), household cooked chichpeas (∆), canned chickpeas (▲), household cooked green lentils (◊), canned brown lentils (♦), household cooked flageolets (+), upper panel) to analyze the relationship between the presence of fibers, phytates, saponins, tannins (dotted arrows, lower panel) and fat-soluble vitamin bioaccessibility (dashed arrows) and uptake (thick arrows).

Figure 4 :

 4 Figure 4 : Effect of specific pulse compounds on vitamin K bioaccessibility.In vitro digestion experiments were performed with test meals containing minced beef meat, refined olive oil supplemented with vitamin K, potatoes, and enriched in purified cellulose (0.7mg), phytates (42.5 mg), saponins (6 mg) and tannins (0.4 mg). Vitamin K bioaccessibility was calculated as the percentage of vitamin recovered in mixed micelles divided by the amount of vitamin recovered in the digesta at the end of the digestion. Data are means ± SEM (n=4 per condition). * p<0.05, ** p<0.01, ** p<0.001 when compared with control.

Figure 5 :

 5 Figure 5: Effect of fibers, phytates, saponins and tannins on t vitamin K uptake by Caco-2 cells

  , E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323

	www.mnf-journal.com	Page 8	Molecular Nutrition & Food Research

  , E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323

	www.mnf-journal.com	Page 13	Molecular Nutrition & Food Research

Table 1 :

 1 Household cooking method

	Pulses	Soaking in low ionized water*	Cooking time in mineral water **
	Kidney beans	Overnight at room temperature;	1h30; ratio seeds: water (w:w) of 1:2.
	White beans	ratio seeds: water (w:w) of 1:5.	1h30; ratio seeds: water (w:w) of 1:2.
	Chickpeas		2h; ratio seeds: water (w:w) of 1:2.
	Green lentils		25 min; ratio seeds: water (w:w) of 1:2.
	Flageolets		1h30; ratio seeds: water (w:w) of 1:2.

* (water hardness: 10-15°F), ** Pyrenea water (Auchan, Croix, France).

Table 2 :

 2 Canning method

	Pulses	Soaking in low ionized water*	Blanching	Canning temperature
	Kidney beans	Overnight at room temperature;	5 min at	16 min at 127 °C, then cooled
	White beans	ratio seeds: water (w:w) of 1:3.	90°C	to 30 °C for 10 min in brine
	Chickpeas Brown lentils			(0.5% salt); ratio seeds: brine (w:w) of 195:236.
	* (water hardness: 10-15°F)

Comment citer ce document : Margier, M., Antoine, T., Siriaco, A., Nowicki, M., Halimi, C., Maillot, M., Georgé, S., Reboul, E. (2019). The Presence of Pulses within a Meal can Alter Fat-Soluble Vitamin Bioavailability. Molecular Nutrition and Food Research, 63 (11), e1801323. , DOI : 10.1002/mnfr.201801323

Molecular Nutrition & Food Research

This article is protected by copyright. All rights reserved.