Fabrice Bationo

Christèle Humblot

Laurencia T Songré-Ouattara

Fatoumata Hama-Ba

Manon Le Merrer

Morgane Chapron

Susanna Kariluoto

Youna M Hemery
email: youna.hemery@ird.fr

Total folate in West African cereal-based fermented foods: bioaccessibility and influence of processing

Keywords: Folate, processing, cereal, digestion, fermentation, Africa, infant food, complementary feeding, food analysis, food composition Simulated gastric fluid SIF: Simulated intestinal fluid SSF: Simulated salivary fluid

Cereal-based fermented foods (CBFF) are frequently consumed by young children in West-Africa. Seven CBFF types (gelatinized doughs, porridges, dumplings, and fritters, made from corn, sorghum or pearl millet) were produced according to the commonly used methods in traditional production units in Burkina Faso. Their total folate content, analyzed using a microbiological method, was between 1.8 and 31.3 µg/100g fresh weight. Folate degradation occurred at several steps during the cereal grain processing into CBFF, for instance during debranning (-51%), soaking (-20% to -49%) and wet-milling (-39% to -59%). Conversely, fermentation increased the folate content of some CBFF (+9 to +27%). Folate bioaccessibility, assessed using a static in vitro digestion model, ranged from 23% to 81%.

The bioaccessible folate content was influenced by the total folate content, the food matrix structure, and folate stability. CBFF contribution to the reference folate intake for young children was at most 23%. This work generated new information on the influence of traditional processing on the vitamin B9 content of CBFF, and the first data on folate bioaccessibility in West African CBFF.

Introduction

Folate (vitamin B9) is essential for human metabolism, because it is involved in cell division, DNA replication, methylation, nucleotide biosynthesis, and amino acid metabolism [START_REF] Mcnulty | Intake and status of folate and related B-vitamins: considerations and challenges in achieving optimal status[END_REF][START_REF] Preedy | Food and Nutritional Components in Focus[END_REF]. Folate deficiency is a global health issue, in both developed and low-income countries [START_REF] Mclean | Review of the magnitude of folate and vitamin B-12 deficiencies worldwide[END_REF][START_REF] Youngblood | Update on global prevention of folic acid-preventable spina bifida and anencephaly[END_REF], and leads to many health disorders, such as neural tube defects, megaloblastic anemia, and colorectal cancer [START_REF] Bailey | Biomarkers of Nutrition for Development-Folate Review[END_REF][START_REF] Fao | Vitamin and mineral requirements in human nutrition[END_REF]. Recent studies carried out in West African countries (Burkina Faso, Ivory Coast, Mali and Senegal) showed that folate deficiency is a severe public health problem among women of reproductive age and children [START_REF] Arsenault | Seasonal differences in food and nutrient intakes among young children and their mothers in rural Burkina Faso[END_REF]Bationo et al., 2019a;[START_REF] Fiorentino | Nutrient Intake Is Insufficient among Senegalese Urban School Children and Adolescents: Results from Two 24 h Recalls in State Primary Schools in Dakar[END_REF][START_REF] Kennedy | Food Groups Associated with a Composite Measure of Probability of Adequate Intake of 11 Micronutrients in the Diets of Women in Urban Mali[END_REF][START_REF] Rohner | Prevalence and public health relevance of micronutrient deficiencies and undernutrition in pre-school children and women of reproductive age in Côte d'Ivoire, West Africa[END_REF]. For instance, in 2010, the folate dietary intake was adequate only in 12% of women of reproductive age in Burkina Faso [START_REF] Becquey | Micronutrient Adequacy of Women's Diet in Urban Burkina Faso Is Low[END_REF].

Cereals are the main crops in Africa (45% of arable land), and make an important contribution to the human diet in this continent, due to the high consumption of cereal-based products [START_REF] Galati | The Cereal Market and the Role of Fermentation in Cereal-Based Food Production in Africa[END_REF]. They cannot be eaten raw, and are processed before consumption.

Fermentation is widely used in many West African countries to transform cereals (sorghum, maize and millet) into edible products [START_REF] Guyot | Cereal-based fermented foods in developing countries: ancient foods for modern research[END_REF]. Traditional cereal-based fermented foods (CBFF), such as porridges or gelatinized doughs, are frequently consumed by young children in West Africa [START_REF] Greffeuille | Traditional recipes of millet-, sorghum-and maize-based dishes and related sauces frequently consumed by young children in Burkina Faso and Benin -European Project INSTAPA[END_REF][START_REF] Hama | Rétention et biodisponiblité du fer et du zinc au cours des procédés de préparation des plats traditionnels à base de céréales locales ou biofortifiées, consommés par les jeunes enfants au Burkina Faso[END_REF]. Fermentation is one of the oldest ways to preserve food products, and to enhance their sanitary and nutritional quality through the production of numerous nutritional and anti-microbial substances by fermenting microorganism [START_REF] Nout | Rich nutrition from the poorest -Cereal fermentations in Africa and Asia[END_REF]. Fermentation is also considered as a good way to increase the folate content of food products, because yeast and many lactic acid bacteria (LAB) can synthesize folate during fermentation [START_REF] Moslehi-Jenabian | Beneficial Effects of Probiotic and Food Borne Yeasts on Human Health[END_REF]Saubade et al., 2017a).

Previous works have reported the isolation of folate-producing strains from different cereals (Cappozi et al., 2012;[START_REF] Salvucci | Technological properties of Lactic acid bacteria isolated from raw cereal material[END_REF]. Moreover, studies on CBFF that are commonly consumed in industrialized countries, such as rye, wheat and oat sourdoughs, have shown that fermentation can improve their folate content [START_REF] Kariluoto | Effect of baking method and fermentation on folate content of rye and wheat breads[END_REF], 2006a[START_REF] Laiño | Development of a high folate concentration yogurt naturally bio-enriched using selected lactic acid bacteria[END_REF][START_REF] Jägerstad | Increasing natural food folates through bioprocessing and biotechnology[END_REF].

Other processing steps used in cereal processing, such as milling, debranning, soaking and cooking, also can influence the folate content of cereal-based foods (Arcot et al., 2002b;Kariluoto et al., 2006b;[START_REF] Witthöft | Folates -food sources, analyses, retention and bioavailability[END_REF]. However, data on the influence of cereal processing on folate content in African cereal-based foods are very scarce. One recent study [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF] assessed the influence of traditional processing steps (soaking, wetmilling, sieving, and fermentation) on the final folate content of a porridge commonly eaten in Burkina Faso. Three other studies have shown that folate can be produced by selected folateproducing microorganisms during fermentation of African maize-based or pearl millet-based porridges [START_REF] Greppi | Ability of lactobacilli isolated from traditional cereal-based fermented food to produce folate in culture media under different growth conditions[END_REF][START_REF] Hjortmo | Production of folates by yeasts in Tanzanian fermented togwa[END_REF]Saubade et al., 2017b).

Moreover, the absorption of dietary folate mainly depends on its bioaccessibility (i.e., the fraction of a nutrient that is released from the food matrix and is available for intestinal absorption) [START_REF] Parada | Food microstructure affects the bioavailability of several nutrients[END_REF]. Folate bioaccessibility can be affected by many factors, including the action of gastrointestinal enzymes and pH during digestion, the presence of inhibitors, and the matrix effects [START_REF] Arkbåge | Bioaccessibility of Folic Acid and (6S)-5-Methyltetrahydrofolate Decreases after the Addition of Folate-Binding Protein to Yogurt as Studied in a Dynamic In Vitro Gastrointestinal Model[END_REF][START_REF] Parada | Food microstructure affects the bioavailability of several nutrients[END_REF][START_REF] Ringling | Simulation of Food Folate Digestion and Bioavailability of an Oxidation Product of 5-Methyltetrahydrofolate[END_REF]. Very few studies have evaluated folate bioaccessibility from different food types, and to our knowledge, none focused on folate bioaccessibility in traditional African cereal-based foods. However, to improve folate intake from African CBFF, more accurate information on traditional cereal processing methods and their influence on folate content is required, as well as on the folate bioaccessibility of these commonly eaten food products.

Therefore, the aim of this study was to provide information on the influence of processing methods of African CBFF on their total folate content and folate bioaccessibility. It also allowed evaluating the potential contribution to the recommended folate intake of young children of these CBFF that are frequently consumed in Burkina Faso and other West African countries (i.e., porridges, fritters, gelatinized doughs, dumplings).

Material and methods

Materials

Plant materials

The cereals used for the production of CBFF were maize (Zea mays), sorghum (Sorghum bicolor), and pearl millet (Pennisetum glaucum). These cereals were purchased by the producers from their usual suppliers at local markets in Ouagadougou (Burkina Faso).

Sample collection

Seven CBFF types were investigated in this study: akassa and doncounou (made from corn), kaffa (made from sorghum), and massa, fura, ben-kida, and ben-saalga (made from pearl millet). Akassa, doncounou and kaffa are gelatinized doughs, massa are fritters, fura are dumplings, and ben-kida, and ben-saalga are porridges. Photographs of these CBFF are presented in Figure 1. These food products were sampled in 20 traditional production units (TPUs) in Ouagadougou, Burkina Faso. The processing method used for the production of each CBFF is described in Figure 2, and in supplementary material.

The processing of ben-saalga, the most commonly eaten CBFF, was monitored in detail at four different TPUs (three different days for each TPU). The parameters implemented at each steps were precisely recorded (e.g., material used, time, temperature, pH, and weight of the different fractions and by-products). Samples of pearl millet grains (raw material), intermediate/final products and by-products were also collected.

For five other CBFF (akassa, doncounou, massa, fura, and ben-kida), samples of raw materials (i.e., grains of pearl millet, or corn) and final products (fermented foods, as eaten by consumers) were collected in three different TPUs for each food type. Moreover, in each TPU, samples were collected on three different days. The process steps were not followed in details, but were listed by recording the producers' declarations, including estimations of the fermentation and cooking times. For kaffa, the least commonly eaten CBFF, samples of raw material (sorghum) and final product were collected on three different days from only one TPU.

For all seven CBFF, in each TPU, three 50g samples were collected per day, on 3 different days (i.e., 9 samples collected per TPU). They were used for dry matter analysis (on site), and then individually stored at -20°C for total folate analysis and in vitro digestion.

Chemicals and enzymes

Porcine pancreatic α-amylase (E-PANAA, #150402, 110.8 U/mg) was purchased from Megazyme (Bray, Ireland). Chicken pancreas γ-glutamylhydrolase (P2002) was obtained from R-Biopharm. Protease from Streptomyces griseus (P5147, 3.5 units/mg), Aspergillus oryzae alpha-amylase (A9857, #SLBM0836V, 50.4 U/mg), pepsin from porcine gastric mucosa (P7000, #BCBF9832V, 536 U/mg), bovine pancreatic trypsin (T0303, #SLBR6593V, 160.6 U/mg) and chymotrypsin (C4129, #SLBG2821V, 40 U/mg), and bovine and ovine bile extracts (B8381, #SLBH1720V, 881.3 g/mol) were purchased from Sigma-Aldrich (St Louis, MI, USA). All the enzymes were stored at -20 °C. All other used chemicals were of analytical grade.

Methods

Ben-saalga mass balance assessment

The traditional processing of pearl millet into ben-saalga has been described before [START_REF] Tou | Study through surveys and fermentation kinetics of the traditional processing of pearl millet (Pennisetum glaucum) into ben-saalga, a fermented gruel from Burkina Faso[END_REF]. To follow the folate losses and gains during ben-saalga processing, and to monitor folate distribution in pearl millet fractions, a mass balance assessment was carried out at each TPU, as described by [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF]. Briefly, the weight of all utensils and sampled products (samples taken for folate analysis, raw materials, intermediate products, by-products, and final products) was recorded at each step of the process. The dry matter (DM) content of all samples was measured, then each sample weight and DM content were combined with the folate content to calculate the amount of folate (in µg) in the original pearl millet grains, and in the intermediate and final products. The folate content distribution in the intermediate and final products was expressed as the percentage of the initial folate quantity in pearl millet grains.

Dry matter content and total lipid content

The DM content of all samples was measured by drying the samples in an air-circulating oven at 105°C in open dishes until constant weight (overnight, ~16h). Samples were then cooled in a desiccator and weighted, and the DM content was expressed as g DM/100 g sample (ICC Standard methods, 2003).

In massa samples, the lipid content also was measured because the frying step led to oil absorption that influenced their DM content. Massa samples were first lyophilized and ground, and then lipids were extracted with diethyl ether using a semi-automatic 2055 Soxtec system (Foss, Nanterre, France), according to the Association of Official Agricultural Chemists Official Methods 2003.05 and 2003.06 (AOAC, 2006). The defatted DM was calculated by subtracting the lipid content from the DM content, and was used to assess the influence of processing on massa folate content, on a dry matter basis.

Determination of total folate content

Total folate content was determined in triplicate using the microbiological assay described by [START_REF] Kariluoto | Total folate[END_REF] and adapted by [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF]. Briefly, thawed samples were diluted in extraction buffer and heated at 100°C for 10 min. After adjusting the pH to 7.2, a tri-enzymatic extraction with α-amylase (20 mg/ml in 1% sodium ascorbate), chicken pancreas γ-glutamylhydrolase (100 mg/ml in Milli-Q water) and protease (3 mg/ml in 1% sodium ascorbate) was used to release folate from the food matrix. After appropriate dilutions of the supernatants, folate concentration was determined in microtiter plates with Lactobacillus rhamnosus ATCC 7469 as the growth indicator organism, folic acid (Sigma-Aldrich F7876) as the calibration standard, and Folic Acid Casei Medium (Difco, Sparks, MD, USA) as the assay medium. The method performance was confirmed by analyzing a certified reference material (BCR 121 Wholemeal flour, IRMM, Geel, Belgium) for each incubation. The certified folate value for this reference material was 500 ± 70 ng/g DM, and in this study, we obtained an average content of 512 ± 57 ng/g dm (n = 38). A blank sample (that contained all the reagents, but no food sample) was also analyzed in each series of samples, and the folate value obtained for the blank sample was deducted from the value obtained for the samples, to take into account the folate brought by the enzymes.

Determination of bioaccessible folate content and folate bioaccessibility

Folate bioaccessibility was assessed using the static in vitro digestion model described by [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF], with minor modifications. This method includes oral (simulated salivary fluid, SSF, pH 7), gastric (simulated gastric fluid, SGF, pH 3), and small intestinal (simulated intestinal fluid, SIF, pH 7) phases (cf. Figure 4 in supplementary material). As the folate content of pancreatin was high, purified enzymes (i.e., trypsin, chymotrypsin, and pancreatic α-amylase) were used. Prior to in vitro digestion, the enzyme activity was checked to guarantee the method efficiency, as described by [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]. After quantifying the folate content of several bile extracts, the one with the lowest folate content (77 ng/g) was chosen, in order not to skew the bioaccessible folate content due to high endogenous content in the bile extracts.

In a 50 ml Falcon tube, 5 g of roughly ground sample was mixed with 4 ml of SSF containing α-amylase, 25 µl of CaCl2 solution and 975 µl of Milli-Q water to reach a final ratio of food/reagents of 50 : 50 (W/ V). The mixture was incubated at 37°C in a shaking water bath for 2 min. Then, 8 ml of SGF with pepsin and 5 µl of CaCl2 solution were added, and the pH was adjusted to 3 with 6 M HCl. After adjusting the volume to 20 ml with Milli-Q water, the mixture was incubated in a shaking water bath at 37°C for 2 hours. To stimulate intestinal digestion, 10 ml of SIF containing bile extract, 6 ml of SIF containing pancreatic α-amylase, 1 ml trypsin solution, 1 ml chymotrypsin solution, and 40 µl of CaCl2 solution were added.

After adjusting the pH to 7 with 2M NaOH, samples were incubated in a shaking water bath at 37°C for 2 hours. Finally, samples were centrifuged at 8000 g at 4°C for 30 min, and supernatants (i.e., the digesta) were stored at -20°C for bioaccessible folate analysis. All samples were digested in duplicates.

The bioaccessible folate content was determined in 4 ml of digesta (i.e., supernatants of the digested samples) that contained the bioaccessible folate, extracted from the food matrix during the in vitro digestion. The folate analysis was carried out as described above, except that only the conjugase (γ-glutamylhydrolase) was added to convert the bioaccessible polyglutamyl folate into the mono and diglutamyl folate forms that were quantified using the microbiological assay.

Bioaccessible folate (in µg/100g fresh weight, FW) corresponds to the folate fraction that is released from the food matrix, and potentially available for absorption through the intestinal barrier. Folate bioaccessibility (in %) was calculated using the following formula: Folate bioaccessibility (%) = Bioaccessible folate/Total folate * 100.

Estimation of CBFF contribution to the folate intake of infants

To estimate the daily folate intake (DFI, in µg/day) from the seven CBFF, known data on the daily frequency (DF) of consumption and portion sizes (PS, in g) of each CBFF for young children aged 6 to 35 months [START_REF] Hama | Rétention et biodisponiblité du fer et du zinc au cours des procédés de préparation des plats traditionnels à base de céréales locales ou biofortifiées, consommés par les jeunes enfants au Burkina Faso[END_REF] were used. Based on these data and on the total folate content (TFC in µg/100g FW) of the seven CBFF, their DFI was calculated as follows: DFI = (TFC/100) * PS * DF For breastfed children, the folate intake from breast milk was also taken into account, based on a folate content of 85 µg/l in breast milk [START_REF] Allen | B Vitamins in Breast Milk: Relative Importance of Maternal Status and Intake, and Effects on Infant Status and function[END_REF], as well as the average daily amount of breast milk consumed by children in developing countries, as described by the World Health Organization (WHO) (1998).

Then, the respective contribution of CBFF and breastmilk to the recommended folate intake (RNI) were calculated, taking into account the specific RNI for each age group (FAO/ [START_REF] Fao | Vitamin and mineral requirements in human nutrition[END_REF]. The DFI needed from other food products (DFI-OFP) than breast milk to reach 100% of the RNI was also calculated. Finally, based on the DFI-OFP, the daily CBFF intake needed to reach 100% of the RNI, if only breast milk and CBFF were consumed, was calculated.

Statistical analysis

Statistical analyses were performed using Statgraphics Centurion 18 (FranceStat, Neuilly, France). The Spearman's rank correlation was used to determine the strength of the relationships between variables. One-way analysis of variance (ANOVA) and Newman-Keuls post-hoc tests were used to identify significant differences between samples. Differences were considered statistically significant with p-values <0.05. Three-level nested ANOVA was also carried out to test the inter-producer and the inter-day (within producers) variabilities. These variables are nested, meaning that each value of these nominal variables (i.e., the subgroups 'producer' and 'day') are found in combination with only one value of the higher-level nominal variable (i.e., the groups 'product' or 'producer').

The linear model for three-way unbalanced nested arrangement is given by: yijkl = μ + αi + βj(i) + γk(ij) + elijk where: yijkl is the l th observation within the k th level of factor 'day' within the j th level of factor 'producer' within the i th level of factor 'product'; μ represents the overall mean; αi is the fixed effect due to the i th level of factor 'product' (i.e., either the type of raw material used, or the type of CBFF produced), with i = 1 to 3 (for raw materials) or i = 1 to 7 (for CBFF); βj(i) is the random effect due to the j th level of factor 'producer' nested within the i th level of factor 'product', with j = 1 to 20; γk(ij) is the random effect due to the k th level of factor 'day' nested within the j th level of factor 'producer' nested within the i th level of factor 'product', with k = 1 to 60; and eijkl is the residual error of the observation yijkl.

Variance components were calculated and expressed as percentages.

Results

Description of food products and processes

The processes used to produce the seven CBFF from different raw materials (corn, sorghum, and pearl millet) are summarized in Figure 2. More details on the description of process steps and food products are provided in the supplementary materials.

Akassa, doncounou, and kaffa are thick fermented and cooked gelatinized doughs that are consumed as staple foods by all age categories, and generally eaten with a sauce. These three CBFF are produced using similar processes. The main difference between akassa and doncounou is that doncounou is made from non-dehulled corn (whole-grain), whereas akassa is made from debranned and degermed corn. Kaffa is made from sorghum. Akassa, doncounou, and kaffa processes included grain soaking, washing, draining, wet milling, sieving, paste fermentation, hydrothermal cooking, and wrapping of gelatinized dough balls in plastic bags. Doncounou processing also included a steam-cooking step.

Massa are fritters (small and thick pancakes fried in oil) made from pearl millet batter, often prepared as street food and eaten as snacks. They are consumed alone or crumbled in porridge. Processing included grain washing and sun drying, before dry milling in a community mill, mixing with water and kneading of the dough, fermentation during settling, and frying. Frying led to oil adsorption in the fritters, with average oil contents of 15.5 ± 4.6, 6.7 ± 2.8, and 17.1 ± 2.4 g/100g FW for massa produced in TPU 1, 2 and 3, respectively.

Fura is a semi-solid dumpling meal made from pearl millet. It can be consumed in different ways. Fura balls can be made into porridge by crumbling and mixing them with porridge, fermented milk, or water with sugar. This can be used as food, refreshing drink, or weaning food for infants. Fura processing included grain dehulling before washing and sun drying (for 30 min for producers P2 and P3, and up to 10 h for P1). After dry milling, sieving was an optional step. Two fermentation steps were carried out: one before and one after cooking.

Ben-saalga and ben-kida are fermented porridges made from pearl millet, very often used as complementary food for infants and young children. Their processes were similar and included the following steps: soaking and washing of the grains, wet-milling, addition of water and sieving, fermentation (during settling), and cooking. For the preparation of benkida, previously prepared lumps (made from pearl-millet flour) were added to the boiling mixture to produce porridge with a granulated texture.

Dry matter content of raw materials and fermented foods

Measurement of the raw material DM content (Table 1) did not highlight any significant difference among cereal grains, whatever the producer or the day of production. Conversely, the DM content of the different CBFF types (Table 1) varied significantly. DM content was very low in porridges (ben-kida and ben-saalga) (5.7 to 8.3 g/100g). The DM contents of the three gelatinized doughs (akassa, doncounou, kaffa) varied greatly (from 11.1 to 30.2 g/100g). The highest DM content (56.7-63.6 g/100g) was observed in fritters (massa). The DM content of doncounou and massa were significantly different among TPUs. A nested analysis of variance showed that for doncounou, this was due to inter-producer variability (88.6% of the variance) rather than inter-day variability (11.0% of the variance). Conversely, for massa, 89.4% of variance was explained by inter-day variability, and only 10.5% by interproducer variability.

The massa DM content (Table 1) included the lipid content brought by oil absorption during frying. The defatted DM content was also calculated, and was 48.1 ± 3.8, 50.0 ± 4.4 and 45.1 ± 5.7 g/100g for massa produced in TPU 1, 2 and 3, respectively.

Folate content of raw materials and fermented foods

Analysis of the folate content of raw materials and fermented foods (Table 1) showed significant differences among cereal types. The folate content of raw corn and sorghum ranged from 13.8 to 21.2 µg/100g FW, whereas it was much higher in pearl millet (54.4 to 73.4 µg/100g FW). The corn grain color did not influence the folate content, because no significant difference was observed between white and yellow corn (15.7 ± 3.0 µg/100g FW and 17.5 ± 2.9 µg/100g FW, respectively). The folate content variability in pearl millet grains was mainly due to inter-producer variability (32.5% of variance) and inter-day variability (26.1% of variance).

Significant differences in total folate content were observed in the seven CBFF. A three-level nested analysis of variance showed that 84.0% of variance was due to inter-food variability, 13.0% to inter-producer variability, and 1.9% to inter-day variability. Folate content was very low in porridges (ben-kida and ben-saalga: 1.8 to 2.9 µg/100g FW), and also in gelatinized doughs (akassa, doncounou, and kaffa: 1.8 to 5.2 µg/100g FW). The highest folate content was observed in dumplings (fura): 16.2 to 31.3 µg/100g FW. Folate content in massa and fura significantly varied from one producer to another. A nested ANOVA showed that for massa this variability was due to both inter-producer and inter-day variability (52.5% and 37.7% of the variance, respectively), whereas for fura it was mostly explained by inter-producer variability (87.8%) rather than inter-day variability (6.4%). Indeed, the folate content of fura produced in TPU 1 (the one that used sun drying instead of draining) was significantly lower than in the other TPUs.

Influence of processing on CBFF folate content

The first step of akassa processing (i.e., corn grain debranning and degerming) resulted in major folate losses because debranned corn contained only 49.6 ± 14.0% of the folate detected in the initial grains (Table 6, in supplementary material). The folate content ranged from 13.8 to 24.3 µg/100g DM in whole-grain corn, and from 5.0 to 12.8 µg/100g DM in debranned corn.

Folate loss ranged from 23% to 66%, but no significant difference among TPUs was observed.

Table 2 summarizes the folate loss or gain in the final food products compared with the raw materials. To avoid the influence of the various water amounts added during processing, the folate content of the different CBFF was expressed in µg/100g DM, and compared with that of cereal grains. The high standard deviations observed for some massa and fura samples were probably due to their variable dry matter contents. The highest folate loss was observed in fritters (massa; reduction between 59% and 76%), followed by porridges (reduction between 44% and 59% for all ben-kida and ben-saalga samples, regardless of the producer).

Conversely, folate content in kaffa was increased by 66%. Folate content was greatly reduced (by 53%) in the dumplings made by the first fura producer, whereas no folate loss was observed in the fura made by the other producers. Likewise, folate content was not significantly different between raw material and CBFF in most akassa and doncounou samples, except for one doncounou producer (folate loss of 22%) and one akassa producer (folate gain of 78%).

Impact of the different ben-saalga processing steps on folate content

The changes in folate content distribution in the samples from the four TPUs (expressed as percentages of the pearl millet folate content, on a dry matter basis) (Figure 3A) indicated that with the exception of producer P2, the soaking step resulted in a significant decrease in folate content (20% to 49% loss, compared with raw pearl millet). This was partly due to folate diffusion in the soaking water (5% to 15%). The folate content in soaked grains varied from 24.2 ± 4.1 µg/100g FW (for P4) to 38.5 ± 10.5 µg/100g FW (for P2). Folate loss in soaking water was higher for producer P2, compared with the other producers, probably due to the longer soaking time (from 24 to 48 h soaking for producer P2, compared with 10 h, 24 h, and 22 h for producers P1, P3 and P4, respectively). After the soaking step, only two producers (P1 and P2) applied a washing step, which did not significantly affect folate content.

Conversely, wet-milling resulted in a significant folate loss, compared with soaked grains, except for producer P4. Indeed, the paste of milled soaked grains contained only 41% to 61% of the folate initially present in pearl millet grains (Figure 3A). The sieving step also led to folate loss due to fractionation (draff removal). Indeed, all sieved batter samples contained significantly less folate than the pastes of milled grains (20% to 37% of the initial pearl millet folate content). The folate content varied from 1.0 ± 0.3 to 4.1 ± 1.4 µg/100g FW in the sieved batter, and from 8.3 ± 2.2 to 11.7 ± 3.2 µg/100g FW in draff. The sum of the amount of folate present in the sieved batter and in draff samples was significantly lower than the total amount of folate in milled grains (except for producer P3). This shows that folate losses also occurred due to other causes. The sieved batter was then left to rest at ambient temperature (decantation and fermentation) for 10 to 13 h (producers P1, P3 and P4) and for 24 h (producer P2). The folate content in the decanted paste (21% to 28% of the initial pearl millet folate) and in supernatant (6% to 18% of the initial pearl millet folate) samples was comparable among producers (Figure 3B). The sum of the folate present in the decanted paste and total supernatant (Decanted paste + TS) samples was significantly higher than the folate fraction in the sieved batter samples for producers P1 and P2. To prepare the final products, producers used only part of the supernatant (used supernatant, US) to dilute the decanted paste and cook the dish (Decanted paste + US) to obtain ben-saalga. Then, ben-saalga was cooked for 5 to 10 min (producers P1 and P4) and for 15 to 20 min (producers P2 and P3). The cooking step did not significantly affect folate distribution. Nevertheless, for all producers, the cooked ben-saalga contained only 21% to 35% of the folate initially present in raw pearl millet grains.

Bioaccessible folate and folate bioaccessibility

Significant differences in bioaccessible folate content (expressed as µg/100g fresh weight) were observed among CBFF types (Table 3). A three-level nested analysis showed that 80.5% of total variance was due to inter-food variability, whereas inter-producer variability and inter-day variability accounted for 7.1% and 10.2% of variance, respectively. Bioaccessible folate was very low (0.4 to 3.2 µg/100g FW) in porridges (ben-kida, ben-saalga) and most of the gelatinized doughs (akassa, doncounou, and kaffa). The highest bioaccessible folate content (4.5 to 8.6 µg/100g FW) was observed in fritters (massa) and dumplings (fura); however, the bioaccessible folate content significantly varied among producers. Nested analyses of variance showed that for fura, 62.5% of the variance was due to inter-producer variability and 18.2% of the variance to inter-day variability. Conversely, for massa, 79.6% of the variance was due to inter-producer variability and only 16.4% to inter-day variability.

Folate bioaccessibility values also varied among the seven studied CBFF. The inter-food variability was the main contributor (73.8%) to the total variance, whereas the inter-producer and inter-day variability only accounted for 7.9% and 9.9% of variance, respectively. Folate bioaccessibility values were low (between 23% and 30%) for akassa, kaffa and fura, and average (between 30% and 57%) for the two porridges (ben-kida, ben-saalga). The highest values (up to 81%) were observed in massa. Folate bioaccessibility was much higher for doncounou (57-67%) than for akassa (24-29%), two gelatinized doughs made of corn.

The bioaccessible folate content was significantly correlated only with the DM content and the total folate content of the fermented foods (r = 0.80 and r = 0.87, respectively).

CBFF contribution to the recommended folate intake of infants

The total folate contents were used to calculate the potential contribution of the seven CBFF to the recommended folate intake of children aged 6-35 months (Table 4). The estimated daily folate intakes from CBFF were 0.8 to 6.4 µg/day, 2.7 to 15.2 µg/day, and 7.9 to 34.6 µg/day, for children aged 6-11 months, 12-23 months, and 24-35 months, respectively. When taking into account the contribution of breast milk to the recommended folate intake for these three age categories, 30%, 69% and 100% of the folate intake should come from sources other than milk. However, the potential contribution of the seven studied CBFF to the recommended DFI was low, from 1% to 8%, 2% to 10%, and 5% to 23% for children aged 6-11 months, 12-23 months, and 24-35 months, respectively. When the bioaccessible folate contents were used to calculate the potential contribution of the seven CBFF to the recommended folate intake of young children, their potential contribution to the recommended DFI was even lower (from 0 to 8%), (Table 5 in supplementary material).

Taking into account the folate intake from breast milk, the daily folate intake that should come from other food sources (DFI-OFP) was 23.9, 103.3, and 150.0 µg/day for children aged 6-11 months, 12-23 months, and 24-35 months, respectively (Table 4). If the DFI-OFP were to come only from cereal-based fermented foods, the amount of the various CBFF that should be consumed to reach 100% of the recommended folate intakes would range from 93-1073 g/day for children aged 6-11 months to 584-6735 g/day for children aged 24-35 months, which would not be realistic.

Discussion

CBFF are staple foods in many African countries [START_REF] Nout | Rich nutrition from the poorest -Cereal fermentations in Africa and Asia[END_REF]. To the best of our knowledge, the influence of traditional processing on the folate content of CBFF commonly eaten in Africa had not been studied yet, except for ben-saalga [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF]. Moreover, folate bioaccessibility in traditional African CBFF has never been investigated. The present study provides new information on CBFF frequently consumed in Burkina Faso. Nevertheless, our findings are also relevant for many other West African countries. Indeed, the analyzed food products are also frequently produced and consumed in Benin and Togo (akassa, doncounou, massa, kaffa, ben-saalga commonly called koko in Benin), Ghana (fura, doncounou, kaffa), Ivory Coast (akassa, massa, ben-saalga and ben-kida commonly called baca or coco baca), Niger (kaffa, doncounou), and Nigeria (fura, massa commonly called wômi) [START_REF] Ayo | Effect of Different Cereals on the Quality of Masa[END_REF][START_REF] Greffeuille | Traditional recipes of millet-, sorghum-and maize-based dishes and related sauces frequently consumed by young children in Burkina Faso and Benin -European Project INSTAPA[END_REF][START_REF] Inyang | Effect of Germination and Fermentation of Pearl Millet on Proximate, Chemical and Sensory Properties of Instant "Fura"-A Nigerian Cereal Food[END_REF][START_REF] Owusu-Kwarteng | Identification of lactic acid bacteria isolated during traditional fura processing in Ghana[END_REF][START_REF] Soro-Yao | Microbiology of Ivorian Fermented Products: A Review[END_REF]. Considerable variations were observed in the folate content of the raw materials. Pearl millet grains displayed the highest folate content, and corn and sorghum the lowest folate content.

The folate content of corn and sorghum was close to that of the West African Food Composition Table (WAFCT), whereas that of pearl millet was much higher than the one reported in the WAFCT (29-31 µg/100g FW) and also in a recent study (Saubade et al., 2017b;[START_REF] Stadlmayr | West African food composition table[END_REF]. This large variation in folate contents may be due to differences in grain cultivars, growing conditions, and storage conditions, as previously reported [START_REF] Edelmann | Folate in barley grain and fractions[END_REF][START_REF] Kariluoto | Effects of Environment and Genotype on Folate Contents in Wheat in the HEALTHGRAIN Diversity Screen †[END_REF]. Moreover, folate content was low in all studied CBFF, irrespective of the food type, producer and starting raw material. Fura exhibited the highest folate content (16 to 31 µg/100g FW), and ben-saalga, ben-kida and akassa the lowest (1.8 to 3.1 µg/100g FW). The difference in folate content between raw material and final food products indicates that part of the initial folate content was lost during food processing to obtain fritters (massa) and porridges (ben-saalga and ben-kida). Conversely, for dumplings (fura), the folate loss during food processing depended on the producer (folate loss observed only for one producer). This suggests that folate was not degraded during fura processing and that the added water only 'diluted' the vitamins initially present in the grains. Alternatively, some loss might have occurred but folate was produced at one stage or another to counterbalance this loss, for example during the fermentation steps. Owusu-Kwarteng et al. ( 2012) described the diversity of LAB species involved in the traditional processing of fura, and some of these LAB might be able to produce folate (Saubade et al., 2017a).

Analysis of the CBFF process steps suggests that some procedures may particularly affect folate content. For instance, debranning performed during akassa processing particularly affected folate content (50% folate loss during this step, in average). This explains the significantly lower folate content in this dish compared with doncounou, another gelatinized dough produced from non-debranned corn grains. Indeed, vitamin B9 is mainly located in the outer layers and germ of cereal grains, and is largely lost during debranning/degerming [START_REF] Liukkonen | Process-induced changes on bioactive compounds in whole grain rye[END_REF][START_REF] Pomeranz | Chemical composition of kernel structures[END_REF]. During grain soaking, folate could have been lost through leaching in soaking water [START_REF] Witthöft | Folates -food sources, analyses, retention and bioavailability[END_REF]. The longer soaking time (24-48 h) used by producer P2 during ben-saalga processing, compared with the other producers (10-24 h soaking), might explain the higher folate content in the P2 soaking water sample. Folates are sensitive to heat, light, and oxidation. Sun drying, which is performed in the TPU yards, also could have affected folate content through the combined action of atmospheric oxygen and light [START_REF] Preedy | Food and Nutritional Components in Focus[END_REF][START_REF] Strandler | Challenges in the Determination of Unsubstituted Food Folates: Impact of Stabilities and Conversions on Analytical Results[END_REF]. This could explain the significantly lower folate content of the fura dumplings produced in the TPU that included a very long sun drying step. Moreover, the CBFF under study were cooked (porridges), fried (fritters), or boiled in water (dumplings, gelatinized doughs). Cooking can lead to thermal and oxidative degradation of folates [START_REF] Delchier | Folates in Fruits and Vegetables: Contents, Processing, and Stability[END_REF][START_REF] Witthöft | Folates -food sources, analyses, retention and bioavailability[END_REF]. Specifically, frying can generate significant folate losses in various food products [START_REF] Aramouni | Dynamics and Bioavailability of Folates in Bovine Liver as a Function of Processing and Frozen Storage[END_REF]Arcot et al., 2002a;[START_REF] Hefni | Folate content in processed legume foods commonly consumed in Egypt[END_REF]. Boiling in water (i.e., hydrothermal cooking) results in significant losses due to folate leaching from the food matrix into the cooking water [START_REF] Delchier | Folates in Fruits and Vegetables: Contents, Processing, and Stability[END_REF][START_REF] Hefni | Folate content in processed legume foods commonly consumed in Egypt[END_REF].

The present work also shows that the folate content of the gelatinized doughs produced in two TPUs (kaffa, and the akassa made by producer P1) was significantly higher than that of their respective raw materials (sorghum and corn), suggesting folate production during the fermentation steps. Indeed, it was previously reported that fermentation positively influences folate content [START_REF] Nout | Rich nutrition from the poorest -Cereal fermentations in Africa and Asia[END_REF], due to the ability of yeast and some LAB to produce folate in fermenting foods [START_REF] Moslehi-Jenabian | Beneficial Effects of Probiotic and Food Borne Yeasts on Human Health[END_REF]Saubade et al., 2017a). However, despite this folate content increase, the final folate content in these ready-to-eat foods was still very low, due to their low DM content. [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF] carried out a mass balance assessment during the processing of pearl millet grains into ben-saalga, but in controlled laboratory conditions. In the present study, although the final ben-saalga folate content was comparable to the 1.5 to 2.5 µg/100g FW reported by [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF], the influence of the different processing steps was not similar. This highlights the difference between analyses in controlled time/temperature conditions and in the yard of TPUs. Specifically, [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF] reported folate production (+26% to +31%) during pearl millet soaking, whereas in the TPUs folate was lost during the soaking step (-19% to -49%). In the TPU yard, grains are left in water at ambient temperature, exposed to sunlight and atmospheric oxygen, until the end of the soaking step [START_REF] Tou | Study through surveys and fermentation kinetics of the traditional processing of pearl millet (Pennisetum glaucum) into ben-saalga, a fermented gruel from Burkina Faso[END_REF]. This can lead to folate oxidization or photo-degradation [START_REF] Delchier | Folates in Fruits and Vegetables: Contents, Processing, and Stability[END_REF][START_REF] Preedy | Food and Nutritional Components in Focus[END_REF]. Moreover, the soaking time was variable (10 to 24h) among TPUs, and might not have been sufficient to promote germination, which leads to folate synthesis (Kariluoto et al., 2006b). [START_REF] Saubade | Influence of fermentation and other processing steps on the folate content of a traditional African cereal-based fermented food[END_REF] found that the fermentation step did not have any significant effect on folate content. Conversely, in the present study, a significant gain of folate was observed during the fermentation step in two TPUs, suggesting folate production by microorganisms present in the batter. Spontaneous fermentation can lead to variable microbial composition (Saubade et al., 2017a) due to the endogenous bacteria present in the cereals, food utensils and manual handling. This could explain the folate content variability in ben-saalga from the different TPUs. In ben-saalga, LAB are the predominant microorganisms during fermentation (Saubade et al., 2017b;[START_REF] Tou | Study through surveys and fermentation kinetics of the traditional processing of pearl millet (Pennisetum glaucum) into ben-saalga, a fermented gruel from Burkina Faso[END_REF]. LAB isolated from ben-saalga display a good genetic potential for folate synthesis, and some specific strains can produce folate during fermentation [START_REF] Greppi | Ability of lactobacilli isolated from traditional cereal-based fermented food to produce folate in culture media under different growth conditions[END_REF]. A recent study also showed that ben-saalga folate content could be increased by using back-slopping or starter cultures combined with amylolytic LAB strains (Bationo et al., 2019b).

Folate bioaccessibility in CBFF was very variable (from 23% to 81%) depending on the food product. Bioaccessibility could have been influenced by i) the total folate content in nondigested foods; ii) the food product structure that governs the digestion patterns of the carbohydrate fractions in cereal foods [START_REF] Lovegrove | Role of polysaccharides in food, digestion, and health[END_REF][START_REF] Mishra | Wholeness and primary and secondary food structure effects on in vitro digestion patterns determine nutritionally distinct carbohydrate fractions in cereal foods[END_REF] and might influence the release of folate from food matrices; and iii) the stability of folate vitamers during the digestion process. [START_REF] Ringling | Simulation of Food Folate Digestion and Bioavailability of an Oxidation Product of 5-Methyltetrahydrofolate[END_REF] also observed a noticeable variability of folate bioaccessibility in various food matrices. They found that this variability was due to differences in the food product composition and in folate vitamer distribution within the food products. Specifically, they observed differences in vitamer stability during in vitro digestion that affected their relative bioaccessibility. For example, tetrahydrofolate was completely lost during the digestion simulations. In the current study, the seven CBFF could be classified in groups that behaved differently during digestion and presented distinct folate bioaccessibility profiles:

• Porridges were characterized by very low bioaccessible folate (< 1.5 µg/100g FW), due to the low total folate content and moderate bioaccessibility ratios (30% to 57%). In these semi-liquid foods, most folate was already in a solubilized form and should have been bioaccessible. However, porridges had little structural resilience in digestion conditions, and folate was not protected by the food matrix during the gastric phase. As many folate vitamers are sensitive to low pH, the observed moderate bioaccessibility ratios could be due to loss of labile folate in acidic gastric conditions [START_REF] Ringling | Simulation of Food Folate Digestion and Bioavailability of an Oxidation Product of 5-Methyltetrahydrofolate[END_REF][START_REF] Seyoum | Properties of Food Folates Determined by Stability and Susceptibility to Intestinal Pteroylpolyglutamate Hydrolase Action[END_REF][START_REF] Strandler | Challenges in the Determination of Unsubstituted Food Folates: Impact of Stabilities and Conversions on Analytical Results[END_REF].

• Gelatinized doughs made from debranned grains (akassa, kaffa) displayed low bioaccessible folate (0.4-1.6 µg/100g FW) and low bioaccessibility (23-30%). Folate bioaccessibility was similarly low in dumplings (fura), but bioaccessible folate was much higher (4-9 µg/100g FW) due to a higher initial total folate content. In these three structurally weak foods, the starchy matrix hydrolysis started during the oral phase of digestion, possibly leading to the degradation of non-protected folate during the gastric phase.

• The two CBFF with the highest folate bioaccessibility ratio were both made from wholegrain cereals, and had either a dense semi-solid structure (doncounou, 57-67% bioaccessibility) or a solid structure (massa, 67-81% bioaccessibility). These dense matrices provided secondary structure and generated particles that were resistant to digestion [START_REF] Mishra | Wholeness and primary and secondary food structure effects on in vitro digestion patterns determine nutritionally distinct carbohydrate fractions in cereal foods[END_REF], and persisted long enough to protect folate from the acidic gastric conditions. Moreover, it is known that micronutrient encapsulation in the plant cell walls modulates the rate and extent of nutrient release during digestion [START_REF] Parada | Food microstructure affects the bioavailability of several nutrients[END_REF]. Therefore, the presence of bran particles in massa and doncounou may have protected folate during the gastric phase, and allowed a higher proportion of folate to reach the intestinal phase (neutral pH) in a non-degraded form.

In many West African countries, traditional millet-, sorghum-and maize-based fermented dishes are frequently consumed by young children [START_REF] Greffeuille | Traditional recipes of millet-, sorghum-and maize-based dishes and related sauces frequently consumed by young children in Burkina Faso and Benin -European Project INSTAPA[END_REF]. Porridges are mainly consumed as foods to complement breast milk. Then, when children can eat family dishes, porridges are progressively replaced by thicker starchy staple foods (such as gelatinized doughs) that are eaten with various sauces. Snacks, such as fritters or dumplings, are given more regularly [START_REF] Hama | Rétention et biodisponiblité du fer et du zinc au cours des procédés de préparation des plats traditionnels à base de céréales locales ou biofortifiées, consommés par les jeunes enfants au Burkina Faso[END_REF]. Our study shows that, whatever the age group, the contribution of the studied CBFF to the recommended folate intake was far from sufficient to cover the folate requirement of young children. By taking into account the food intake reported by Hama (2012), 6-11-month-, 12-23-month-and 24-35-month-old children would get respectively 1-8%, 2-10% and 5-23% of the recommended folate intake through CBFF consumption. Moreover, the potential contribution of the seven studied CBFF to the recommended DFI was limited: up to 23% when considering the total folate contents, and only up to 8% when considering the bioaccessible folate contents. This suggests that many children might be at risk of folate deficiency, especially non-breastfed children, if their diet were mainly based on CBFF. However, in Burkina Faso and other West African countries, most of these CBFF are consumed with other food products with higher folate contents, such as sauces made from green leafy vegetables and legumes (e.g., groundnut or soybean) [START_REF] Greffeuille | Traditional recipes of millet-, sorghum-and maize-based dishes and related sauces frequently consumed by young children in Burkina Faso and Benin -European Project INSTAPA[END_REF].

Conclusion

This work generated new information on the influence of traditional processing on the folate content and the first data on folate bioaccessibility in seven CBFF commonly consumed in West Africa that can be useful to complete the existing food composition tables. The total folate and bioaccessible folate contents of the seven studied products (gelatinized doughs, porridges, dumplings, fritters) were rather low, suggesting that populations whose diet is mainly based on these CBFF may be at risk of folate deficiency. The folate content of most of the food products was strongly and negatively affected by the laborious processing. Some processing steps could be adapted to limit such losses (controlled volumes, controlled soaking and fermentation times, and limitation of direct exposure to sunlight). Another way to increase the folate content in CBFF could be to promote folate production during the fermentation step, by using folate-producing bacterial strains. Finally, the observed differences in folate bioaccessibility ratios among the studied food products suggest a strong effect of the food matrix that should be investigated more precisely. The influence of processing on folate bioaccessibility in other folate-rich foods frequently consumed by young children together with CBFF (i.e., green leafy vegetables, legumes) should also be analyzed. Bationo F., Humblot C., Songré Ouattara L.T., Hama Ba F., Le Merrer M., Chapron M., Kariluoto S., Hemery Y., 2019. Total Results are the mean ± standard deviations (samples collected on three different days for each producer, and analyzed in triplicate). *: Significant difference of folate content between raw material and processed fermented food product (ANOVA, p < 0.05). a : For massa (fritters made from pearl millet batter), the defatted dry matter was used for the calculation, to take into account the absorption of oil during frying. a Contribution of CBFF to RNI calculated using the daily intakes and daily intake frequencies reported by [START_REF] Hama | Rétention et biodisponiblité du fer et du zinc au cours des procédés de préparation des plats traditionnels à base de céréales locales ou biofortifiées, consommés par les jeunes enfants au Burkina Faso[END_REF] for various CBFF, and for the indicated age groups. b RNI values, as reported by FAO/WHO (2004). c Daily intake of breast milk as reported by [START_REF] Who | Complementary feeding of young children in developing countries, A review of current scientific knowledge[END_REF]. d Folate intake from breast milk based on a folate content of 85 µg/L in breast milk [START_REF] Allen | B Vitamins in Breast Milk: Relative Importance of Maternal Status and Intake, and Effects on Infant Status and function[END_REF]). e Folate intake needed from other food products than breast milk to reach the RNI. f Daily CBBF intake that would be needed if only breast milk and CBFF were consumed.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Processing of cereal-based fermented foods in traditional processing units. "Yes"

Figure 3 .

 3 Figure 3. Changes in folate content distribution during the steps of the traditional processing

Flow diagram of a simulated in vitro digestion method adapted from the method described by Minekus et al. (2014). Table 1. Dry matter content and total folate content in raw materials and fermented foods Food products (and respective raw materials) Producer Dry matter content (g/100g FW) Total folate content (µg/100g FW) Raw materials Fermented foods Raw materials Fermented foods

  folate in West-African cereal-based fermented foods: bioaccessibility and influence of processing. Journal of Food Composition and Analysis.

	5g sample + Simulated		Oral phase		Digesta
	salivary fluids + α-amylase + Simulated gastric fluids + pepsin + Sim. intestinal fluids + bile extract + purified enzymes (trypsin, chymotrypsin, pancreatic α-amylase) (gelatinized dough, corn) P1 P2 P3 Doncounou (gelatinized dough, corn) P1 P2 P3 Kaffa (gelatinized dough, sorghum) P1 Massa* (batter fritters, pearl-millet) P1 P2 P3 Fura P1 Figure 4. Akassa (dumplings, P2	5 min at 37°C Gastric phase (pH 3) 2h at 37°C Intestinal phase (ph 7) 2h at 37°C 89.7 ± 0.5 89.9 ± 0.1 89.6 ± 0.4 88.4 ± 0.7 88.8 ± 0.6 90.4 ± 2.4 88.9 ± 0.4 89.4 ± 0.7 89.8 ± 0.8 89.9 ± 0.4 89.6 ± 0.3 89.7 ± 0.8	Bioaccessible folate Extraction (with conjugase only) and microbiological assay of total folate centrifugation supernatant 11.4 ± 1.1 b 13.8 ± 1.3 j 11.1 ± 0.2 b 16.8 ± 1.3 jk 13.0 ± 1.1 b 21.2 ± 1.4 k 24.1 ± 0.9 d 15.7 ± 2.4 j 26.6 ± 1.6 e 14.8 ± 2.0 j 30.2 ± 0.1 f 13.8 ± 1.7 j 17.4 ± 3.1 c 16.3 ± 2.6 jk 63.6 ± 8.1 i 57.2 ± 3.7 l 56.7 ± 3.8 h 72.6 ± 4.0 n 62.2 ± 6.2 i 63.3 ± 3.9 lm 42.5 ± 1.9 g 73.2 ± 3.9 n 42.2 ± 3.1 g 65.4 ± 3.7 mn	6 3.1 ± 0.8 op 1.8 ± 0.3 o 2.7 ± 0.4 o 4.7 ± 0.8 p 4.8 ± 0.6 p 3.6 ± 0.6 p 5.2 ± 1.3 pq 7.2 ± 1.3 q 10.2 ± 1.0 qr 12.6 ± 3.2 rs 16.2 ± 1.2 s 31.3 ± 2.5 t
	pearl-millet)	P3	89.1 ± 1.0	42.6 ± 1.2 g	64.0 ± 9.7 mn	29.6 ± 4.1 t
	Ben-kida	P1	90.0 ± 0.6	8.3 ± 0.9 a	54.4 ± 4.2 l	2.5 ± 0.4 o
	(porridge, pearl-	P2	90.5 ± 0.7	6.1 ± 0.7 a	65.3 ± 7.0 mn	2.5 ± 0.3 o
	millet)	P3	90.4 ± 0.5	7.9 ± 0.7 a	59.3 ± 6.9 l	2.5 ± 0.8 o
	Ben-saalga (porridge, pearl-millet)	P1 P2 P3 P4	89.0 ± 0.7 90.0 ± 0.3 89.3 ± 0.9 89.2 ± 0.1	5.8 ± 0.4 a 6.6 ± 1.9 a 5.7 ± 0.6 a 6.6 ± 0.1 a	62.6 ± 6.3 lm 70.3 ± 6.9 mn 68.6 ± 7.4 mn 73.4 ± 8.2 n	1.9 ± 0.3 o 2.9 ± 1.3 op 1.8 ± 0.2 o 2.4 ± 1.0 o
	Results are the mean ± standard deviations (for each producer, samples were collected on three
	different days, and analyzed in triplicate). ANOVA: In each column, values with no common letter
	are considered significantly different (p<0.05). No significant differences were observed for the
	dry matter content of raw materials. *For massa samples, the results are given in g or µg /100g
	FW of food "as eaten" (i.e., non-defatted samples).

Table 2 . Folate loss or gain during the processing of cereal grains into fermented foods

 2

	Food products (and raw materials)	Producer	Folate content (µg/100g DM) Raw material Processed food	Folate loss or gain (%)
	Akassa	P1	15.4 ± 1.5	27.5 ± 6.2	+78% *
	(gelatinized dough,	P2	18.7 ± 1.4	16.6 ± 3.0	-11%
	corn)	P3	23.6 ± 1.5	20.6 ± 4.1	-13%
	Doncounou	P1	17.8 ± 2.8	19.6 ± 3.5	10%
	(gelatinized dough,	P2	16.7 ± 2.2	17.9 ± 1.8	7%
	corn)	P3	15.2 ± 1.9	11.8 ± 1.9	-22% *
	Kaffa
	(gelatinized dough,	P1	18.3 ± 2.9	30.4 ± 7.3	+66% *
	sorghum)
	Massa	P1	63.9 ± 3.9	15.1 ± 3.3	-76% *
	(batter fritters,	P2	80.8 ± 4.5	20.5 ± 1.8	-75% *
	pearl-millet) a	P3	70.4 ± 4.4	28.9 ± 9.6	-59% *
	Fura	P1	81.7 ± 4.3	38.1 ± 3.5	-53% *
	(dumplings,	P2	72.9 ± 4.1	74.5 ± 6.9	2%
	pearl-millet)	P3	71.9 ± 11.2	69.7 ± 10.4	-3%
	Ben-kida	P1	60.5 ± 4.4	30.8 ± 5.8	-49% *
	(porridge,	P2	72.1 ± 7.6	40.4 ± 4.1	-44% *
	pearl-millet)	P3	65.7 ± 7.8	32.0 ± 1.2	-51% *
	Ben-saalga (porridge, pearl-millet)	P1 P2 P3 P4	70.3 ± 7.4 78.1 ± 7.8 76.9 ± 8.1 82.3 ± 9.3	32.3 ± 7.2 41.5 ± 9.5 31.8 ± 5.2 36.2 ± 1.5	-54% * -47% * -59% * -56% *

Table 3 . Bioaccessible folate and folate bioaccessibility in fermented food products

 3 Results are the mean ± standard deviations (samples collected on three different days for each producer, and analyzed in quadruplicate). ANOVA: in each column, values without common letters are considered significantly different (p<0.05).

	Food product	Producer	Bioaccessible folate (µg/100g FW)	Folate bioaccessibility (%)
	Akassa	P1	0.9 ± 0.4 a	29.1 ± 6.8 i
	(gelatinized dough,	P2	0.4 ± 0.1 a	23.6 ± 5.4 i
	corn)	P3	0.7 ± 0.2 a	26.4 ± 6.2 i
	Doncounou	P1	3.2 ± 0.8 c	66.9 ± 10.9 n
	(gelatinized dough,	P2	2.8 ± 0.5 c	57.4 ± 7.6 lm
	corn)	P3	2.2 ± 0.5 bc	62.4 ± 11.1 mn
	Kaffa
	(gelatinized dough,	P1	1.6 ± 0.4 ab	29.8 ± 4.5 i
	sorghum)
	Massa	P1	5.6 ± 0.5 e	77.6 ± 5.8 o
	(batter fritters,	P2	8.3 ± 1.3 fg	80.9 ± 7.7 o
	pearl-millet)	P3	8.6 ± 3.2 g	66.8 ± 13.3 n
	Fura	P1	4.5 ± 0.8 d	27.7 ± 4.9 i
	(dumplings,	P2	7.4 ± 1.5 f	23.5 ± 4.9 i
	pearl-millet)	P3	8.2 ± 1.6 fg	27.8 ± 3.9 i
	Ben-kida	P1	1.1 ± 0.2 ab	45.5 ± 10.7 jk
	(porridge,	P2	1.1 ± 0.2 ab	43.5 ± 9.0 j
	pearl-millet)	P3	1.4 ± 0.6 ab	55.7 ± 10.8 lm
	Ben-saalga (porridge, pearl-millet)	P1 P2 P3 P4	1.1 ± 0.3 ab 1.2 ± 0.4 ab 1.0 ± 0.2 a 0.6 ± 0.1 a	57.3 ± 7.5 lm 45.7 ± 8.1 jk 52.9 ± 10.9 kl 30.1 ± 8.8 i
	Folate bioaccessibility (%) = Bioaccessible folate / Total folate * 100

Table 4 . Potential contribution of West-African cereal-based fermented foods to the recommended folate intakes for children aged 6-35 months

 4

		Daily	CBFF	Recommended	Daily	Daily	Contribution	Folate	CBFF
		CBFF	contribution	folate intake	breast	folate	of breast	intake	intake
		folate	to the RNI a	(RNI) b	milk	intake	milk to RNI	from foods	needed
		intake			intake c	from		to reach	to reach
						breast		100%	100%
						milk d		RNI e	RNI f
		(µg/day) (% of RNI)	(µg/day)	(ml/day) (µg/day) (% of RNI) (µg/day) (g/day)
	6-11 months (breastfed)
	Akassa	3.2	4%						940
	Doncounou	2.5	3%						549
	Kaffa	4.6	6%						456
	Massa	0.8	1%	80	660	56.1	70%	23.9	239
	Fura	1.4	2%						93
	Ben-kida	6.2	8%						961
	Ben-saalga	6.4	8%						1073
	12-23 months (breastfed)
	Akassa	10.8	7%						4066
	Doncounou	8.2	5%						2372
	Kaffa	15.2	10%						1973
	Massa	2.7	2%	150	550	46.7	31%	103.3	1032
	Fura	4.3	3%						402
	Benk-ida	4.8	3%						4154
	Ben-saalga	5.0	3%						4640
	24-35 months (non-breastfed)
	Akassa	24.6	16%						5902
	Doncounou 18.7	12%						3443
	Kaffa	34.6	23%						2864
	Massa	8.2	5%	150	0	0	0%	150.0	1499
	Fura	14.0	9%						584
	Ben-kida	7.9	5%						6029
	Ben-saalga	8.2	5%						6735

Acknowledgements

This work was carried out in the framework of the FolEA project that is part of the ERA-Net «Developing African-European joint collaboration for Science and Technology» (ERAfrica), financially supported by the European Commission under the 7th Framework Programme.

The authors would like to thank Christian Picq for the analysis of crude lipids, Sonia Fortin for advices on statistical methods, Edwige Oboulbiga Bahanla for her help with the collection of food samples in Ouagadougou, and Dounia Gueye and Nelly Fioroni for their appreciated help with the folate analyses and in vitro digestions.


Tanguy Davin

Benoit Lefez

Alain Guillet

S Berbescu

M Matt

A Dinh

S Simeon

L Noussair

L Armand-Lefevre

B Davido

Supercooling of phase change: A new modeling formulation using apparent specific heat capacity

Keywords: meningococcal bacteremia, purpura fulminans, doxycycline

Case presentation

An 18-year old Caucasian male, belonging to the group of men having sex with men (MSM), was admitted to the hospital due to fever (40°C), headaches and arthromyalgia that evolved with an erythematous rash.

The recent patient history revealed that 2 weeks ago he visited the South of France (French Riviera) with his parents, doing trips in the forest and collecting trash. Then he returned to Paris to complete his high school graduation, followed by a couple of days of wild camping with friends. While camping he was bitten by a tick on the back 72 hours before his admission.

The day after the tick bite he noticed an erythematous and purpuric rash (Figure 1) on his legs and pectoral area that was associated with fever and cephalalgia. Within 24 hours he presented an unusual fatigue, diarrhea, nausea and vomiting. At the Emergency Room, the clinical examination revealed a febrile patient, at 40°C, with hypotension (80/50 mmHg) and a generalized non pruritic macular rash that disappeared in vitropression. There were no neurological abnormalities.

Blood test showed elevated C reactive protein (150 mg/L) and neutrophilic leukocytosis, absence of renal dysfunction and normal transaminase values. Serologies for hepatitis C and B viruses, syphilis and HIV were negative.

In the hypothesis of a bacterial infection, antimicrobial therapy by 1 dose of amoxicillinclavulanate of 875/125 mg was administered intravenously and the patient was transferred to our department without clinical improvement the day after.

The cerebrospinal fluid (CSF) was clear and colorless. The CSF cell count was 2/mm 3 without red blood cells, hyperglycorachia (4.7 mmol/L) and normoproteinorrachia (0.22 G/L; reference value 0.20-0.40 G/L).

During the first hours of hospitalization, the appearance of the typical rash and the presence of tick bite history, made us suspicious of rickettsiosis and the patient got started on oral doxycycline 200 mg daily with rapidly a resolution of symptoms within 24 hours after his admission.

Blood culture at 48 hours came back positive for Neisseria meningitidis serotype B.

An antimicrobial susceptibility testing was performed following the guidelines of the European

Committee of Antimicrobial Susceptibility Testing (EUCAST) and showed that the strain was susceptible to all antibiotic tests, with the exception of a reduced susceptibility to amoxicillin that also applies to amoxicillin-clavulanate: MIC (minimum inhibitory concentration) > 0.5 μg/ml). In addition MIC to other antibiotics were as follows: cefotaxime (MIC <0.002 μg/ml), tetracycline (MIC 0.19 μg/ml), minocycline (minimum inhibitory concentration <0.064 μg/ml), tigecycline, ciprofloxacin and rifampicin.

Considering clinical and biological resolution under treatment (decrease from 18,000 WBC and a CRP level at 137 mg/L at day 0 to 4800 WBC and a CRP level at 10 mg/L at day 5), doxycycline was continued for a total of 5 days. Thereafter the patient was discharged with one dose of intravenous Ceftriaxone 1g administered for the eradication of pharyngeal carriage.

The patient was followed-up in consultation after one month for vaccination against other serotypes and further investigations revealed no complementary deficiency. He was considered cured with no sign of infection and normal biology. The patient declared the absence of other cases of infection in his close surroundings.

Discussion

In this case report, the atypical clinical presentation associated with the recent history of tick bite, was evocative of a potential rickettsial disease, considering the purpuric rash. So, it was decided to treat the patient with doxycycline. By luck, patient's strain was susceptible to cyclins which allowed full recovery.

Neisseria meningitidis is a gram-negative coccus acquired in the nasopharynx through respiratory spread. The most common manifestations are related to meningitis or bacteremia, but also it might result in localized infections such as arthritis or endocarditis (1,2).

One of the clinical manifestations of the meningococcal bacteremia is purpura fulminans, a rare syndrome of intravascular thrombosis which occurs due to both infectious and non-infectious causes. Infectious causes include meningococcal, streptococcal and staphylococcal sepsis, but the most frequent is meningococcemia [START_REF] Kravitz | Purpura fulminans due to Staphylococcus aureus[END_REF]. Viral and non-infectious etiologies were also described (primary varicella, HHV6 infection, deficiency of protein S or C).

The meningococcal disease is a life-threatening, but vaccine-preventable disease. The serogroups A, B, C, W, X and Y are responsible for the majority of meningococcal disease. In United States, the serogroup B caused 38% of all bacteremia included 69% of cases aged 18-23 years (4).

Using an alternative regimen to ceftriaxone, can be helpful in particular situations, notably in patients allergic to beta-lactams. Even if ciprofloxacin is deemed to be effective, the resistance of Neisseria group to fluoroquinolones was increased in the last decades, from 10% to almost 100% resistance, [START_REF] Yang | Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolated from Shanghai[END_REF][START_REF] Goold | No way back for quinolones in the treatment of gonorrhea[END_REF] particularly in men having sex with men (MSM). This led to other therapeutic alternatives including aztreonam (7). However, aztreonam's spectrum activity does not include gram-positive cocci such as Streptococcus spp. which is why using it can be very dangerous when dealing with a purpura fulminans. Also, in case of unclear anamnesis, meningococcal infection can be misdiagnosed such as illustrated in our case, and doxycycline might find its place in such conditions. Doxycycline has been proposed for decades in the prevention of bacterial meningococcal meningitis and therefore is known to be effective against Neisseria spp. However, the molecule is not usually tested in routine antibiograms, which in our case could be helpful. Moreover, doxycycline in MSM is a common regimen well validated and prescribed against STIs, especially in post-exposure prophylaxis (8). In the present case the patient was tested for Mycoplasma spp. and Neisseria spp. even though the urinary sample was tested after he received ceftriaxone and doxycycline, both came back negative. He was also negative for syphilis and HIV. Doxycycline is a lipophilic bacteriostatic antibiotic, which is completely absorbed after oral administration but not recommended for the treatment of bacteremia, but could be considered in an association. Our patient received an oral antibiotic, 2 times daily, for a total of 5 days.

Treating successfully bacteremia with bacteriostatic antibiotics was already described in staphylococcal infections, but using Tigecycline, a derivate from doxycycline, in intravenous administration (9).

Strains resistant to penicillin have been reported from a large number of countries and in

France the last report stated that 30% of Neisseria meningitidis are resistant to amoxicillin (10).

Overall our own experience describes an original alternative to treat Neisseria species.

The way to prevent morbidity and mortality following meningococcal disease is by vaccination.

Today the vaccinal recommendation includes the vaccine against A, C, W and Y serotype of Neisseria meningitidis. Our patient was infected by a B serotype and currently there is still no broad vaccine available covering all serotype B strains.

Acknowledgments:

SB was in charge of conceptualization, data curation, methodology, project administration, resources and writing -original draft; MM supervised the work; AD visualized the manuscript; SS helped for the methods; LN gave resources for the case; LA helped for resources and visualization; BD supervised, validated and wrote definite version of the manuscript.

Availability of data and materials:

All material and data described in the manuscript are available upon request to the corresponding author of the present article.

This case report highlights the risk of misdiagnosis an authentic meningococcal disease presenting as a rickettsiosis. To the best of our knowledge this is the first case of meningococcal bacteremia treated successfully by oral doxycycline. Our case emphasizes that doxycycline could be an appropriate alternative therapy to intravenous ceftriaxone for particular nonsevere situation or allergy to beta-lactams after susceptibility testing.

Consent for publication:

Written informed consent was obtained from the patient for publication of this case report.

A copy of the written consent is available for review by the Editor of this journal.

Ethics approval and consent to participate:

Not applicable.

Conflict of interest: none.


Anaïs Augias
email: anais.augias@yahoo.fr

Philippe Sansonetti

Philippe Charlier

REFLEXIONS BIO-BANQUE : APPLIQUER LE MODELE MEDICAL A L'ANTHROPOLOGIE Constat et réflexion autour des interventions sur les vestiges humains archéologiques et ethnographiques BIO-BANK: APPLYING THE MEDICAL MODEL TO ANTHROPOLOGY Observation and reflection on interventions on archaeological and ethnographic human remains

Keywords: Anthropologie, Archéologie, Ethique, Intégrité, Loi, Restes humains Anthropology, Archaeology, Ethics, Integrity, Law, Human remains

Les préoccupations scientifiques autour des interventions sur les restes humains en contexte archéologique et ethnographique sont de plus en plus présentes. Il apparaît un réel désir international de statuer et de réfléchir à une transparence et une coalition des états et organismes de recherche dans l'intérêt de ces êtres, de leurs produits biologiques et des populations associées. Les nouvelles technologies, les outils interactifs puis les textes existants offrent une possibilité de réflexion, de réponse dans la manière de gérer, stocker et traiter ces ressources pour lutter contre l'isolement et les inconduites scientifiques face à ces vestiges devenus de simples objets tout en respectant leur caractère humain, leur nature archéologique et ethnographique puis leur potentiel scientifique pluridisciplinaire.

La scène scientifique internationale est animée ces dernières années par un désir d'application plus visible du statut de bien commun des vestiges anthropologiques issus des collections muséales, ethnographiques et archéologiques. Avec l'arrivée de nouvelles technologies toujours plus performantes, ces supports se retrouvent au coeur d'une réflexion éthique quant à leur stockage et leur étude. Le développement des études pluridisciplinaires et le constat d'un isolement général appelle à revoir ces interventions de manière plus intègre tout en respectant la fragilité des ressources, la société associée et leur caractère humain.

Il s'agit de proposer un moyen de répondre à ces préoccupations à partir de textes légaux et d'outils déjà existants, notamment en lien avec l'environnement médical, lequel offre un cadre protecteur plus strict. Il faut penser un lieu virtuel commun et participatif où se rencontrent les vestiges et les acteurs scientifiques en toute transparence pour faciliter l'échange des échantillons et des données stockés, permettre leur diffusion, leur valorisation et le croisement des données pour lutter contre l'accumulation isolée de ces supports et de leurs analyses.

Isolement et anthropo-responsabilité

Ces dernières années, les études interdisciplinaires se multiplient, et notamment sur du matériel biologique humain actuel comme ancien. Ces derniers prennent, peu à peu, une place prépondérante sur l'étude du contexte sanitaire actuel. En effet, au-delà du rôle jusqu'à présent joué dans la compréhension de l'évolution morphologique et géographique humaine, les supports humains archéologiques et ethnographiques (Figure 1) sont de plus en plus impliqués dans le domaine de la santé [START_REF] Charlier | Faeces vivos docent. Microbiome intestinal ancien et problématiques médicales contemporaines[END_REF][START_REF] Charlier | Reemerging infectious disease from the past: Hysteria or real risk?[END_REF][START_REF] Schnorr | Insights into human evolution from ancient and contemporary microbiome studies[END_REF][START_REF] Jersie-Christensen | Quantitative metaproteomics of medieval dental calculus reveals individual oral health status[END_REF]. Les nouvelles biotechnologies appliquées à ces vestiges permettent, par exemple, de confronter les données microbiologiques anciennes et contemporaines (issues de contextes géographiques et chronologiques distincts) afin de mieux comprendre la genèse, l'évolution, la disparition et la résurgence de pathologies ou les raisons de dysbioses. La médecine étant animée par une dynamique d'innovation perpétuelle répondant aux pathologies d'un instant T et l'archéo-anthropologie par une démarche de recherche originelle et évolutive [START_REF] Demoule | L'avenir du passé : modernité de l'archéologie[END_REF], impliquer cette dernière dans les problématiques médicales représente un atout dans la tentative de mise en place de théories générales, convoitées par toute science [START_REF] Demoule | Théories et interprétations en archéologie[END_REF]. Cette démarche offrirait un recul et une diversité nécessaires et une possibilité de comprendre les réponses biologiques à des faits intrinsèques ou extrinsèques, repérer un mécanisme/des régularités [START_REF] Demoule | Théories et interprétations en archéologie[END_REF] à travers le Temps et l'espace, et ainsi anticiper-prévoir-prédire.

Ces échantillons anciens et/ou de cultures contemporaines traditionnelles sont des archives humaines fragiles et des ressources limitées qu'il faut protéger. Ce matériel humain collecté (lors de missions archéologiques ou ethnographiques) est stocké dans les différents organismes de recherche ayant procédé au prélèvement et/ou à l'étude. Cela représente un nombre conséquent d'échantillons humains bruts, c'est-à-dire le prélèvement initial, ou d'échantillons intermédiaires, c'est-à-dire résultant d'une manipulation biologique1 ainsi que des données résultant de l'étude (brutes, publiées ou non). Ces supports vont être entreposés, dans le meilleur des cas, ou jetés. Lorsqu'ils sont conservés, les échantillons sont confinés et non diffusés : chaque organisme a alors sa zone de stockage propre. Ces collections indépendantes ne sont connues que des chercheurs de l'unité en question et sont accessibles à certains spécialistes extérieurs qui en font la demande, parfois, de manière hasardeuse.

Mais, ces vestiges "ne peuvent envahir sans limite des dépôts de fouilles ou musées"

Le temps de l'oubli : disparition statutaire

Le cadre légal français relatif au prélèvement, au consentement, à la conservation et au traitement des supports biologiques humains, en contexte hospitalier et médico-légal, est clairement encadré [START_REF] Dupont | conserver et utiliser des échantillons biologiques humains à l'hôpital[END_REF], notamment avec des textes tels que la loi dite Jardé [START_REF] Loi Dite | 300 du 5 mars 2012 relative aux recherches impliquant la personne humaine[END_REF] Ce dispositif international permettra ainsi des études croisées et diachroniques à partir de milliers de données relatives à l'Homme et un recul chronologique important qu'une étude isolée ne pourra jamais mettre en place puis le développement de nouveaux champs d'études en appliquant, par exemple, les nouvelles découvertes médicales aux supports archéologiques, comme dernièrement l'observation de l'impact du microbiome sur la santé osseuse [START_REF] Hathaway-Schrader | Antibiotic perturbation of gut microbiota dysregulates osteoimmune cross talk in postpubertal skeletal development[END_REF].

Surtout cette démarche rendrait un statut et une considération à ces sujets et échantillons vulnérables et non-pérennes. Pour parvenir à un tel résultat, il est nécessaire de statuer de manière claire sur ces échantillons. Cette proposition pourrait répondre aux attentes visant à améliorer la transdisciplinarité tout en limitant les coûts, la dégradation et le stockage indépendant de biens humains, l'isolement des données et les inconduites.

[ 7 ]

 7 . De ces entrepôts indépendants et isolés puis du manque de centralisation découlent des problèmes non anticipés, allant à l'encontre de l'éthique et facilitant les formes d'inconduites scientifiques, à savoir : -un isolement scientifique et la multiplicité des prélèvements : par le manque d'outil de diffusion, les échantillons humains disponibles et les analyses pratiquées restent confinés et obligent les scientifiques non informés à refaire un même prélèvement et/ou une même analyse, -une forme de commercialisation : les sujets, en contexte ethnographiques, à force de sollicitations, demandent des rémunérations, de même que les inventeurs dans les cas archéologiques particuliers, -une sorte d'appropriation/privatisation du matériel biologique humain par les organismes de recherche et une forme de concurrence. Il s'agit de réfléchir sur l'intégration de nouveaux réflexes scientifiques, d'élargir la protection légale des substances biologiques aux échantillons ethnographiques et archéologiques puis d'offrir un lieu commun d'interaction.

  relative aux recherches impliquant la personne humaine, l'article 1121-1 du Code de la santé publique, les directives 2004/23/CE du 31 mars 2004 du Parlement européen et du Conseil, n°2006/17/CE du 8 février 2006 de la Commission des Communautés européennes, n° 2006/86/CE du 24 octobre 2006 de la Commission des Communautés européennes, etc. De plus, les textes relatifs au corps humain et au respect dû à son statut, notamment avec l'apport de la bioéthique, sont précis : -l'article 225-17 du Code Pénal stipule que : « Toute atteinte à l'intégrité du cadavre, par quelque moyen que ce soit, est punie d'un an d'emprisonnement et de 15 000 euros d'amende. La violation ou la profanation, par quelque moyen que ce soit, de tombeaux, de sépultures, d'urnes cinéraires ou de monuments édifiés à la mémoire des morts est punie d'un an d'emprisonnement et de 15 000 euros d'amende » [10] ; -l'article 16 de la loi n°94-653 du 29 juillet 1994 relative au respect du corps humain : « Le corps humain, ses éléments et ses produits ne peuvent faire l'objet d'un droit patrimonial. Il ne peut être porté atteinte à l'intégrité du corps humain qu'en cas de nécessité thérapeutique pour la personne. » [11]. -la loi du 19 décembre 2008 avec l'article 16-1-1 du Code Civil « Le respect dû au corps humain ne cesse pas avec la mort. Les restes des personnes décédées, y compris les cendres de celles dont le corps a donné lieu à crémation, doivent être traités avec respect, dignité et décence ». En parallèle, les textes relatifs à l'archéologie permettent la protection des sols et des vestiges anciens, dans le cadre préventif comme programmé, notamment par le biais du Code du Patrimoine [12]. Celui-ci traite de la responsabilité de l'Etat, de la circulation, de la vente et de la restitution des biens culturels, la mise en oeuvre des interventions archéologiques, etc. Toutefois, aucune distinction explicite n'est faite entre les biens biologiques humains et les autres vestiges mobiliers. Ainsi, les vestiges humains archéologiques, par les articles 541-4 et 541-5, pourraient encore faire l'objet d'un droit patrimonial, par exemple [13]. Malgré la nature identique des ressources médicales, archéologiques et ethnographiques, la protection est différentielle et plusieurs réflexions, parfois contradictoires, émergent, dues au manque de clarté. La médecine, au-delà de sa réquisition pour le soin ou l'autopsie (déterminer les causes de décès, identifier l'individu et préciser le mécanisme de la mort), n'a légalement pas autorisation à pratiquer des prélèvements ou toute activité de recherche sur le corps du patient. En effet, « l'utilisation des éléments et des produits du corps humain » [14], hors du contexte restreint défini par ces mêmes lois, pourrait relever de l'« atteinte à l'intégrité du cadavre» [10]. Mais toutes ces lois ne mentionnent pas les êtres archéologiques ou ethnographiques. Puisque, dans les textes et les moeurs, ces vestiges humains sont vus comme du simple mobilier et que les demandes d'échantillons humains en contexte médical pour des recherches scientifiques, hors « nécessité thérapeutique pour la personne » [11], suivent un protocole strict, les disciplines médicales peuvent alors utiliser ces supports humains, non autorisées dans leur domaine, pour répondre à des hypothèses de mécaniques humaines, comme un éventuel « moyen thérapeutique indirect ». Au contraire, bien que depuis les années 50, l'intervention sur les inhumations archéologiques ne soit plus considérée comme une violation de sépultures due à son caractère scientifique, l'exhumation et l'étude de son contenu pourrait toujours relever, dans les textes, d'une illégalité tolérée. Cette inconsidération peut alors être en lien avec le décès ancien du sujet et de ses proches, la prescription criminelle [15], la disparition des caractéristiques humaines (tissu mou par exemple), le caractère de don d'échantillons par les populations et le temps de l'oubli grandissant. La question d'une potentielle perte d'humanité des échantillons contemporains issus de dons ou de la personne humaine archéologique se pose. Pour un Centre de Ressources Biologiques (CRB) Si les échantillons biologiques humains en contexte hospitalier et médico-légal sont légalement encadrés et protégés, ce n'est pas le cas des échantillons archéologiques et ethnographiques, qui n'apparaissent dans aucun texte de manières explicite. Pourtant, il s'agit de collections de même nature et leur étude utilise les mêmes outils pour des problématiques similaires. De ce fait, les êtres et produits biologiques humains issus de recherches anthropologiques devraient bénéficier du même statut que leurs homologues biomédicaux. L'application de la loi Jardé [9] à ces supports serait alors un moyen adéquat de protéger ces vestiges fragiles, lesquels sont d'une importance majeure évidente dans les problématiques humaines anciennes, actuelles et futures. La démarche d'élargir le modèle protecteur biomédical à ces archives humaines impliquerait une union et de nouveaux réflexes scientifique comme une éventuelle déclaration de « détention de restes ou produits biologiques humains archéologiques ou ethnographiques à but scientifique », l'enregistrement du projet de recherche impliquant une personne ou une ressource humaine et la soumission du projet à un comité d'expertise scientifique et/ou de protection des personnes (similairement à la loi Jardé). Il est important de réfléchir à une coalition juridique et scientifique afin de ne négliger ni le caractère humain, ni la nature archéologique et ethnographique, ni le potentiel scientifique pluridisciplinaire de ces vestiges. En parallèle de la protection des personnes archéologiques et traditionnelles puis de l'encadrement de leurs études, le stockage et le besoin de communication entre les organismes scientifiques concernant les échantillons disponibles et les données résultantes, dans le but de limiter les prélèvements, les inconvenances et les coûts, préoccupent également la scène scientifique. Ce sentiment est d'autant plus problématique lorsqu'il s'agit de matériel humain, lequel est une archive fragile d'informations pour les problématiques présentes comme futures. L'union des sciences relatives à l'Homme serait bénéfique aux échantillons ainsi qu'à chacune des disciplines concernées (archéologie, anthropologie, biologie, microbiologie, muséologie, ethnologie, médecine, médecine-légale, physique-chimie, toxicologie, radiographique, dentaire, etc.) et nécessite la création d'un lieu commun : un Centre de Ressources Biologiques. Ce dispositif est la norme dans le contexte médical, dont l'objectif est pluriel [16] : collecter, stocker et redistribuer des échantillons biologiques et des données associées de manière strictement réglementée [17]. Toutefois, à l'image de la loi Jardé, il s'agit d'un processus entre parties contractantes. Dans notre cas, il ne faut pas oublier que la démarche originelle du sujet est purement altruiste dans le cas des populations actuelles et non consentie pour les cas archéologiques. Le manque de bio-banques archéo-anthropologiques traduit également une vision différentielle, voire égocentrée, dans laquelle les échantillons actuels sont précieux pour la compréhension et le traitement des populations actuelles et futures mais les ressources du passé ne sont pas considérées comme pouvant jouer le même rôle. Pourtant, ces vestiges biologiques sont exploitables, notamment avec l'essor des biotechnologies [18], et pourraient également, en plus des lois précédemment citées, être concernées par les divers textes et réflexions relatifs à la diversité biologique. Ainsi, tout support osseux, organique, sédimentaire, issus de collections archéologiques, ethnologiques, muséales ou n'entrant plus dans le cadre judiciaire par la prescription criminelle [15], et en accord avec les lois [9, 10], pourrait être concerné. Ces préoccupations sont internationales et la réponse devrait être de même. Pour le soutien et la mise en place d'un tel projet, il est important : -de revoir certains textes, précédemment mentionnés, notamment en distinguant clairement les vestiges biologiques humains archéologiques et ethnographiques dans le Livre V du Code du Patrimoine [12] et dans la loi Jardé [9], lesquels se citeront mutuellement, -et de mobiliser les instances mondiales compétentes tels que : o l'Etat, lequel deviendra le propriétaire exclusif de ces vestiges dans les années à venir [13], et le Ministère de la Culture et de la Communication, par le biais des directions régionales des affaires culturelles (DRAC), o la mission de recherche Droit et Justice qui « est un groupement d'intérêt public (GIP) créé, à l'initiative conjointe du Ministère de la Justice et du CNRS, par un arrêté du 11 février 1994 » [19] o l'Organisation de Coopération et de Développement Economiques (OCDE) qui réunit 30 démocraties, o l'ONU qui agit pour la protection des biens culturels et la science, avec par exemple la Convention sur la Diversité Biologique [20] et le Protocole de Nagoya, o la Convention sur les Droits de l'Homme et la biomédecine (STE n° 164). Conclusion La question des vestiges biologiques des personnes anciennes et traditionnelles s'impose sur le devant de la scène scientifique actuelle et expose la nécessité d'une coalition scientifique et juridique dans le but d'instaurer une transparence des entrepôts puis la circulation et le partage contrôlés et protégés des échantillons et des données associées.

Figure 1 -

 1 Figure 1 -Echantillons et analyses possibles sur un corps humain en contexte archéologique

Figure 1 -

 1 Figure 1 -Samples and possible analyzes on a human body in archaeological and

Ce sont, par exemple, des échantillons conservés lors de certaines manipulations de laboratoire, lesquels peuvent être utiles pour d'autres problématiques. Par exemple, dans le cadre d'une étude sur la bactérie Yersinia pestis, l'étape microbiologique appelée « Enrichment » sépare l'ADN de l'agent responsable de la peste de celui des autres bactéries. Ainsi, deux échantillons sont produits : d'un côté celui contenant la peste, celui sur lequel le spécialiste se penchera pour sa problématique, et de l'autre le reste des bactéries.

Remerciements

Les auteurs remercient Céline Bon (plateau "Paléogénomique et Génétique Moléculaire" du Muséum National d'Histoire Naturelle sur le site du Musée de l'Homme -UMR7206 Eco-anthropologie et Ethnobiologie. Département Hommes, Natures et Sociétés. Musée de l'Homme) pour la relecture de cet article.