Fabien Dumetz

Corinne Buré

Serge Alfos

Marc Bonneu

Emmanuel Richard

Katia Touyarot

Anaïs Marie

Jean-Marie Schmitter

Clémentine Bosch-Bouju

Véronique Pallet
email: veronique.pallet@enscbp.fr

Normalization of hippocampal retinoic acid level corrects age-related memory deficits in rats

Keywords: aging, retinol, retinoic acid, memory, neuronal morphology, hippocampus vit.A, vitamin A, RA, retinoic acid, IU : International Unit, RBPR2, Retinol Binding Protein Receptor 2, LRAT, Lecithin:Retinol AcylTransferase, REH, Retinyl Ester Hydrolase

Dietary micronutrients constitute a major environmental factor influencing aging processes.

Vitamin A (vit. A) is the precursor of retinoic acid, a bioactive molecule that controls the expression of several genes involved in brain function. Evidence suggest a reduction of vit. A bioavailability with aging, but its impact on neuronal network is poorly understood. We investigated the mechanisms linking memory impairments with specific alterations of retinoic acid metabolism in the hippocampus. We compared young (10 weeks) and aged (16 months) rats, supplemented or not with dietary vitamin A (20 IU retinol/g) for 4 weeks. Our study reveals that aging induced dysregulation of gene expression involved in vit. A and retinoic acid metabolism in the liver. Furthermore, vit. A supplementation restored the integrity of the hippocampal neuronal morphology altered by aging. Importantly, we found a high correlation between hippocampal levels of retinoic acid and memory performance. The present work establishes the link between collapse of retinoid metabolism and age-related cognitive decline, highlighting the role of vit. A in maintaining memory through aging.

INTRODUCTION

Mechanisms of aging and age-related cognitive decline are currently a matter of intense research aiming at defining strategies for maintaining a good cognitive state and quality of life of the elderly. At the cellular and network levels, it is now well established that age-related cognitive impairments are paralleled by a loss of synaptic plasticity in the hippocampus, a key structure for memory processes [START_REF] Burke | Neural plasticity in the ageing brain[END_REF][START_REF] Rosenzweig | Impact of aging on hippocampal function: plasticity, network dynamics, and cognition[END_REF][START_REF] Shetty | Chelation of hippocampal zinc enhances long-term potentiation and synaptic tagging/capture in CA1 pyramidal neurons of aged rats: implications to aging and memory[END_REF].

Optimal nutrition appears as a promising way to prevent and slow down age-related cognitive decline, without heavy pharmacological intervention. Indeed, it is now recognized that dietary factors, and micronutrients in particular, can act as signaling molecules to maintain brain functions [START_REF] Gómez-Pinilla | Brain foods: the effects of nutrients on brain function[END_REF]. Among them, vitamin A is of particular interest to maintain cognition along aging [START_REF] Feart | Aging affects the retinoic acid and the triiodothyronine nuclear receptor mRNA expression in human peripheral blood mononuclear cells[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. Vitamin A (vit. A) is a micronutrient provided by animal foods, such as meat and dairy products. It can also be synthesized from carotenoids present in vegetables. In the organism, retinol is metabolized in retinoic acid (RA), its main bioactive derivative, which is implicated in the regulation of a large panel of genes [START_REF] Blomhoff | Overview of retinoid metabolism and function[END_REF]. RA plays crucial roles during development but is also essential at adult age [START_REF] Olson | Significance of vitamin A to brain function, behavior and learning[END_REF][START_REF] Shearer | A vitamin for the brain[END_REF][START_REF] Stoney | A Vitamin on the Mind: New Discoveries on Control of the Brain by Vitamin A[END_REF]. In the brain, RA is notably involved in synaptic plasticity in the hippocampus [START_REF] Aoto | Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity[END_REF]Arendt et al., 2015a;[START_REF] Chen | Synaptic retinoic acid signaling and homeostatic synaptic plasticity[END_REF][START_REF] Misner | Vitamin A deprivation results in reversible loss of hippocampal long-term synaptic plasticity[END_REF].

With aging, vit. A seems to accumulate in the liver, its storage organ [START_REF] Pallet | Aging decreases retinoic acid and triiodothyronine nuclear expression in rat liver: exogenous retinol and retinoic acid differentially modulate this decreased expression[END_REF][START_REF] Van Der Loo | Age-related changes of vitamin A status[END_REF][START_REF] Azaïs-Braesco | Vitamin A status in the institutionalized elderly. Critical analysis of four evaluation criteria: dietary vitamin A intake, serum retinol, relative dose-response test (RDR) and impression cytology with transfer (ICT)[END_REF]; however, its bioavailability for target tissues appears altered. Supporting this, we previously demonstrated that receptors, transporters and molecules involved in the signaling pathway of RA in the brain are dysregulated with aging [START_REF] Enderlin | Age-related decreases in mRNA for brain nuclear receptors and target genes are reversed by retinoic acid treatment[END_REF][START_REF] Pallet | Aging decreases retinoic acid and triiodothyronine nuclear expression in rat liver: exogenous retinol and retinoic acid differentially modulate this decreased expression[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. The resulting decrease in RA signaling is associated with impairments of memory performance. In particular, RA signaling in the brain is crucial for spatial and episodic-like memory [START_REF] Cocco | Vitamin A deficiency produces spatial learning and memory impairment in rats[END_REF][START_REF] Etchamendy | Vitamin A deficiency and relational memory deficit in adult mice: relationships with changes in brain retinoid signalling[END_REF][START_REF] Mingaud | Retinoid hyposignaling contributes to aging-related decline in hippocampal function in short-term/working memory organization and long-term declarative memory encoding in mice[END_REF][START_REF] Olson | Significance of vitamin A to brain function, behavior and learning[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. Recently, an association between low level of circulating retinol and age-related cognitive decline has also been found in humans [START_REF] Huang | Diminished circulating retinol and elevated α-TOH/retinol ratio predict an increased risk of cognitive decline in aging Chinese adults, especially in subjects with ApoE2 or ApoE4 genotype[END_REF].

As a clue of the causality between RA deficiency and memory impairments, vit. A supplementation in aged rodents was shown to reduce memory impairments [START_REF] Mingaud | Retinoid hyposignaling contributes to aging-related decline in hippocampal function in short-term/working memory organization and long-term declarative memory encoding in mice[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. However, mechanisms linking alteration of vit.A metabolism in the liver and hyposignaling of RA pathway in the brain have not been elucidated yet. Additionally, the impact on neuronal networks of a reduced brain concentration of RA is poorly understood. Thus, there is no direct evidence for the relationship between the collapse of vit. A metabolism /RA brain bioavailability and age-related cognitive impairment.

In this study we investigated the mechanisms linking specific alterations of RA metabolism in the hippocampus to memory impairments in aged rats supplemented or not with vit. A. Aging was associated with reduced RA availability and dysregulation of vit. A and RA metabolism in the liver.

Importantly, we found a high correlation between hippocampal levels of RA and memory performance. Vit. A supplementation led to a concomitant restoration of the neuronal morphology of pyramidal cells in the hippocampus and of memory capacities altered with aging.

The present work establishes the link between retinoid metabolic collapse and age-related cognitive decline. It highlights the potential capacity of vit. A supplementation to restore memory capacities. This study brings new and useful knowledge on the importance of RA signaling for cognitive processes during aging.

MATERIAL AND METHODS

Animals and diet

All experiments were performed in accordance to criteria of the European Communities Council Directive 2010/63/UE and the French National Committee .

Experiments were performed on male Wistar rats obtained from Janvier Labs (France). Young (10 weeks) and old (16 months) rats were maintained under standard housing conditions in a temperature-(23 ± 1 °C) and humidity-(40 %) controlled animal room with a 12-h light/dark cycle (0700-1900 hours), with ad libitum access to food and water. Rats were housed 2 per cage and were weighted weekly.

All rats received a custom diet upon arrival for 4 weeks. Control rats were fed control diet, with 5 IU retinol/g (INRA, Jouy-en-Josas), while rats enriched with vit. A received a diet containing 20 IU retinol/g (INRA, Jouy-en-Josas). The two diets were isocaloric and identical except the amount of retinol.

Behavioral assessment (Y-maze test)

Spatial memory was assessed in rats after 4 weeks of dietary protocol. The Y-maze paradigm was used to assess spatial memory as previously described [START_REF] Delpech | Transgenic increase in n-3/n-6 fatty acid ratio protects against cognitive deficits induced by an immune challenge through decrease of neuroinflammation[END_REF]. Rats were handled daily for 1 week before the test. All tests were conducted in a sound-attenuated separate experimental room. Behavioral sessions were recorded with a ceiling-mounted video camera and analyzed using tracking software (ViewPoint Behavioral Technology). The apparatus was a Yshaped maze made of dark grey plastic. Each arm was 34-cm long, 8-cm wide and 14-cm high. The floor of the maze was covered with used litter from the home cages of all animals and was mixed between sessions to scramble olfactory cues. Visual cues were placed in the testing room and kept constant during the whole test. In the first trial of the test, one arm of the Y-maze was closed with a guillotine door and rats were allowed to visit two arms of the Y-maze for 5 min. Closed arms were randomly assigned for each rat. After 2h of inter-trial interval, rats were placed back in the start arm and allowed free access to the three arms for 5 min. Data are presented as the time spent exploring the novel arm during the 5 min of the second trial compared to familiar and start arms.

Vitamin A concentration measurement

Blood collected during euthanasia was spun at 1,500 g for 15 min, the supernatant was removed and stored at -20°C until used. Aliquots of liver collected during euthanasia were stored at -80°C.

Before retinol extraction they were homogenized in sodium phosphate-EDTA (0.05 M, pH 7.8) buffer. Retinol was extracted with hexane/BHT and assayed by HPLC according to a previously described method [START_REF] Biesalski | Rapid determination of retinol (vitamin A) in serum by high pressure liquid chromatography (HPLC)[END_REF].

RA levels measurements

Tissues samples were collected and weighed under red light and retinoids were handled with glass single-use containers and pipettes. RA was extracted by a two-step acid-base extraction as described previously by [START_REF] Kane | Quantification of endogenous retinoids[END_REF]. 5 µM Acitretin (Sigma-Aldrich) was added as internal standard into samples to normalize extraction efficiency. LC-ESI-MS/MS (MRM mode) analyses were performed with a mass spectrometer model QTRAP® 5500 (Sciex, Villebon sur Yvette, France) coupled to a LC system (LC-20AD XR pump (Shimatzu, Marne-la-Vallée, France) and PAL HTC-xt Autosampler (CTC Analytics, Zwingen, Switzerland)). Extracts were dissolved in 35 μL of CH3CN/H2O 80/20. Analyses were achieved in the positive mode; nitrogen was used for the curtain gas (set to 20), gas1 (set to 35), gas2 (set to 0) and as collision gas. Needle voltage was at +5,500 V without needle heating; the declustering potential was set at +101 V and +236 V and the collision energy at +27 eV and +35 eV for RA and acitretin, respectively. MS/MS experiments were performed by following two MRM transitions for RA (301.1/123.2 and 301.1/161.1) and acitretin (327.2/131.0 and 327.2/159.1). One transition is used for quantitation and the other for confirmation. The area of LC peaks was determined using MultiQuant software (v2.1, Sciex). Reversed phase separations were carried out on an Ascentis RP Amide 150×1 mm column, with 3 μm particles (Supelco, Sigma Aldrich, St Quentin Fallavier, France). Eluent A was H2O+0.1 % formic acid and eluent B was CH3CN+0.1 % formic acid. The gradient elution program was: 0 min, 30 % B; 8 min, 30 % B; 10 min, 70 % B; 35-36 min, 87 % B; 37 min, 30 % B. The flow rate was 50 μL/min; 10μL sample volumes were injected. This protocol allows All-trans RA separation from its isomers. For each experiment, a standard curve consisting of triplicated extracted all-Trans RA (Sigma-Aldrich) samples of known concentrations (0.07-2.5 µM) was used to correlate LC peaks area onto RA concentrations.

Neuronal morphology analysis

Golgi-Cox staining. Rats were deeply anesthetized with isoflurane and then decapitated. Four animals per group were used. Brains were processed according to the Golgi-Cox kit guidelines (PK401 FD Rapid Golgi Stain KIT, Neurotechnologies INC, Paris, France) as previously described (Janthakin et al., 2017). Briefly, one hemisphere per brain was immersed in the Golgi-Cox solution for 8 days before deep freezing in isopentane. 100 μm coronal sections containing the dorsal hippocampus were collected at -24 °C using a cryostat (Leica, Solms, Germany) and mounted on 3% gelatin-coated slides. When slides were totally dry (3 days after), they were stained and coverslipped with Depex. Exposure to light was limited during the whole process.

Structural analysis.

Images were obtained at the Bordeaux Imaging Center (CNRS-INSERM and Bordeaux University, France BioImaging) with Nanozoomer slide scanner (Hamamatsu Nanozoomer 2.0 HT) and analyzed using Imaris software (Bitplane, Oxford Instrument Compagny).

The experimenter remained blind to the treatment conditions throughout the procedure.

Dendritic arbor complexity was evaluated in pyramidal neurons of the hippocampal CA1 subfield.

The following criteria were used to select pyramidal neurons: (a) full impregnation of the neurons along the entire length of the dendritic tree; (b) dendrites without significant truncation of branches; and (c) relative isolation from neighboring impregnated neurons, astrocytes or blood vessels. For each rat, 4-6 neurons fulfilling the criteria were finally selected. Dendritic morphology. Briefly, NDPI images at X 20 magnification (one image every 2 μm in the z axis) obtained with Nanozoomer were converted into TIFF format using the ImageJ software (http://imagej.nih.gov.gate2.inist.fr/ij/) and NDPI tools plugin [START_REF] Deroulers | Analyzing huge pathology images with open source software[END_REF]. For each selected neuron, all branches of the dendritic tree were semi-automatically reconstructed in 3D using the Imaris software (Oxford Instruments, Zürich, Switzerland).

mRNA expression analysis by RT-qPCR

Aliquots of liver stored at -80°C were homogenized in 1ml of Trizol reagent (Invitrogen, France) and total RNAs were extracted according to the manufacturer's instructions. The quality and the concentration of the purified RNA was measured by using a Nanodrop One (Ozyme, France). The integrity of RNA samples was assessed using the RNA 6000 Nano LabChip kit in combination with the 2100 Bioanalyzer (Agilent Technologies, France). Using oligo dT and random primers, cDNA was synthesized with ImProm II reverse transcriptase (Promega, France) according to the manufacturer's instructions. The real-time PCR was performed using the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany) with a 384-well format, in a final volume of 10 µl, using the SYBR Green I Master kit, as previously described [START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. The forward and reverse primer sequences used are the following: Rbpr2, 5'-TGCTTCTCATCGGAGGCATG-3' and 5'-

AGTAACCACAAACCAGGTCAGG-3';

Lrat 5'-AGTGTCACGGACCCATTTTACC-3' and 5'-ACCTTCTGAGTGCGTTCCTTG-3'; Reh 5'-TGAAGTTCTGGGCCAACTTTGC-3' and 5'-TGGCACCAATCTGCAAATACCC-3'; Raldh2 5'-AAGCTTGCAGACTTGGTGGAAC-3' and 5'-AAGCTTGCAGGAATGGCTTACC-3'; Cyp26a1 5'-AAGCGCAGGAAATACGGCTTC-3' and 5'-AAGATGCGCCGCACATTATCC-3'; Bmg 5'-GCCCAACTTCCTCAACTGCTACG-3' and 5'-GCATATACATCGGTCTCGGTGGG-3'. The specificity of the amplified products was verified by the melting curve analysis showing a single melting peak after amplification. Data analysis was performed using the Roche's E-method of relative quantification, which uses standard curve derived efficiencies, of the LightCycler 480 1.5 version software. In this study we used the b2microglobulin (BMG) housekeeping gene as the reference gene since its expression level was unaffected by our experimental conditions.

Statistical analysis

Statistical tests were performed with GraphPad Prism 7.0 (GraphPad software, San Diego, CA, USA) using a critical probability of p<0.

RESULTS

RA levels are restored by dietary vit. A in aged rats

Young and 16-month old rats were fed with either control (5 IU retinol/g) or vit. A supplemented diet (20 IU retinol/g) for 4 weeks. We first assessed the levels of retinol and RA in the serum.

Quantitative measurements relied on a purposely designed LC-ESI-MS/MS method using Multiple Reaction Monitoring (MRM). In accordance with previous reports [START_REF] Chevalier | Dietary restriction alters retinol and retinol-binding protein metabolism in aging rats[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF], a lower retinol concentration was found in the serum of aged rats compared to control rats (young: 1.87 ± 0.01 µM vs aged: 0.72 ± 0.02 µM, Figure 1A). Here, we found additionally that RA concentration was also significantly reduced in the serum of aged rats compared to young rats (young: 11.88 ± 3.42 pmol/ml vs aged: 0.92 ± 0.17 pmol/ml, Figure 1B).

These results support a decreased bioavailability of retinol and RA with aging. Of note, individual variability within a group was high for RA concentration, while it was lower for retinol. Four weeks of dietary supplementation with vit. A corrected level of both retinol and RA in the serum of aged rats, but did not alter concentrations in young rats (Figure 1 A, B).

We then quantified levels of RA in the hippocampus, a brain structure highly involved in spatial memory in rats. We found that hippocampal concentration of RA in aged animals was highly decreased compared to young rats (young: 113.9 ± 17.6 pmol/g vs aged: 23.2 ± 5.5 pmol/g, Figure 1C). Furthermore, 4 weeks of vit. A supplementation efficiently normalized RA concentration in their hippocampus (Figure 1C). Of note, hippocampus level of RA correlated with RA level in the serum in aged animals, but not with retinol level (Figure 1D, RA: Spearman r = 0.7614, p = 0.0002; retinol: Spearman r = 0.3973, p = 0.1025). This suggests that blood RA is more likely the source of hippocampal RA.

Metabolism of retinol and RA is altered with aging and restored by supplementation

We hypothesized that decreased levels of retinol and RA observed upon aging originated from a metabolism dysfunction. To investigate this, we compared retinol and RA concentrations in the liver of young and aged rats. We found that retinol and RA concentrations were massively increased in the liver of aged rats compared to young rats (retinol: young: 0.98 ± 0.01 pmol/g vs aged: 5.27 ± 0.47 pmol/g; RA: young: 142.0 ± 9.5 pmol/g vs aged: 422.6 ± 104.1 pmol/g, Figure 2A, B). Notably, Vit. A supplementation for 4 weeks further increased retinol levels in the liver in both young and aged rats (Figure 2A), while RA levels were not affected by vit. A supplementation (Figure 2B).

The discrepancy between decreased levels of retinol / RA in the serum and elevated levels in the liver suggests that aging induces a dysregulation of retinol / RA release from the liver to the serum. To better understand this, we analyzed the mRNA expression level of genes coding for proteins involved in the metabolism of retinol and RA. In aged rats, the gene expression for the protein responsible of retinol entry in the liver, RBPR2 (Retinol Binding Protein Receptor 2) was increased (young: 0.51 ± 0.04 vs aged: 1.02 ± 0.10, Figure 2C). Meanwhile, we observed a decreased mRNA expression for Lrat (Lecithin:Retinol AcylTransferase) (young: 0.83 ± 0.08 vs aged: 0.14 ± 0.05) and a trend to decrease for Reh (Retinyl Ester Hydrolase) (young: 0.33 ± 0.01 vs aged: 0.25 ± 0.03) in the liver of aged rats, suggesting a reduced retinol esterification (Figure 2 D,E). This is in accordance with high levels of retinol in the liver of aged rats. Expression levels of Rbpr2, Lrat and Reh mRNAs were normalized by vit. A supplementation in aged rats. Additionally, the mRNA expression of Raldh2, the enzyme of RA synthesis, was slightly decreased in aged rats compared to young rats, and was significantly increased by vit. A supplementation (young: 0.30 ± 0.03; aged: 0.17 ± 0.06; young + vit. A: 0.23 ± 0.02; aged + vit. A: 0.46 ± 0.05; Figure 2F). Finally, the gene expression of Cyp26a1, coding for the degradation enzyme of RA, was significantly decreased in aged rats and normalized after 4 weeks of vit. A supplementation, similarly to serum levels of RA and retinol (young: 0.40 ± 0.07; aged: 0.14 ± 0.02; young + vit. A: 0.35 ± 0.04; aged + vit. A: 0.40 ± 0.06; Figure 2G).

Together, these data are coherent with a decreased ability of the liver to release retinol and RA with aging. In addition, the regulation of enzymes by the supplement of Vit. A brought by the enriched diet, may contribute to reactivate the release of retinol and RA from liver, and to normalize RA levels in the serum.

Neuronal morphology in aged rats is normalized by vit. A supplementation

Aging is associated to a reduction of hippocampal volume and neuronal arborization in both humans and rodents that is thought to participate to age-related cognitive decline [START_REF] Bartsch | The hippocampus in aging and disease: From plasticity to vulnerability[END_REF][START_REF] Burke | Neural plasticity in the ageing brain[END_REF][START_REF] De Flores | Structural imaging of hippocampal subfields in healthy aging and Alzheimer's disease[END_REF]. To decipher the impact of vitamin A on neuronal network, we analyzed the neuronal morphology of pyramidal cells in the CA1 region of the dorsal hippocampus (Figure 3A). Golgi staining revealed that the total dendrite length of pyramidal neurons was significantly lowered in aged animals compared to young rats; Figure 3 A-C). This reduction in dendrite length was present at both basal (young: 3358 ± 291 µm vs aged: 10 2079 ± 174 µm) and apical (young: 2577 ± 156 µm vs aged: 1738 ± 114 µm) poles of CA1 pyramidal neurons. In details, dendritic lengths between aged and young rats were similar for primary and secondary dendrites, but were reduced for aged rats at higher levels of dendrites (Figure 3 D,E).

This result indicates an atrophy of neurons at more distal dendrites, for both basal and apical dendrite trees. The number of branches was also significantly reduced in aged rats (Apical: young:

41.2 ± 2.7 µm vs aged: 24.9 ± 1.7 µm; basal: young: 53.5 ± 4.9 µm vs aged: 27.4 ± 2.6 µm; Figure 3F, G), indicating a reduction in dendrite ramifications. Sholl intersections were significantly fewer in aged rats at a distance of 40-160 µm from the soma for apical dendrites (Figure 3H) and 60-220 µm from the soma for basal dendrites (Figure 3I).

For aged rats, a diet supplemented in Vit. A ameliorates all measured parameters of both apical and basal dendrite morphology. Total dendrite length of CA1 pyramidal neurons in vit. A supplemented aged-rats was similar to young rats (Figure 3 A-E), as well as the number of branches (Figure 3F, G) and Sholl intersections (Figure 3 H,I). In parallel, Vit. A supplementation in young rats did not alter the measured morphological parameters. Thus, vit. A supplementation abolished alterations of neuronal morphology due to aging.

Spatial memory performance of aged rats match with hippocampal RA levels

It is commonly assumed that dendritic tree structure shapes brain networks and thus influences memory performance. To understand the consequence of RA bioavailability on age-related cognitive decline, we evaluated memory performance with the Y-maze paradigm. The total distance traveled in the Y-maze was not different between the 4 groups (Figure 4A). In the Y-maze task, young rats spent significantly more time in the new arm than in the familiar arm (70.4 ± 2.7 % of time in the new arm; Figure 4B). Conversely, aged rats did not discriminate the familiar arm from new one (42.0 ± 4.7 % of time in the new arm; Figure 4B). Four weeks of vit. A supplementation efficiently corrected impairment of spatial memory observed in aged rats and had no impact on spatial memory of young rats (young + vit. A: 81.5 ± 2.4 % of time in the new arm vs aged + vit. A: 64.1 ± 6.4 % of time in the new arm; Figure 4C).

To clarify the link between hippocampal RA levels and memory, we plotted the time spent in the new arm as a factor of hippocampus RA concentration (Figure 4D). We observed a significant correlation between memory performance and hippocampus RA concentration when collating all groups (Pearson r = 0.6719, p < 0.0001). Conversely, memory performance correlated poorly with serum levels of RA (Spearman r = 0.3172, p = 0.0523).

When groups were considered independently, correlation between memory performance and RA level in the hippocampus was significant only for aged rats (Pearson r = 0.6448, p = 0.0441), and not for young rats (Pearson r = 0.0.3671, p = 0.1340) or aged rats submitted to a diet enriched with vit. A (Pearson r = 0.5242, p = 0.1199) (Figure 4D). While RA hippocampus level seems to become a limiting factor in aged rats, enriched aged rats and young +/-vit. A supplementation exhibited a concentration sufficient to enable good memory performance. We used a non-linear regression to fit optimally the data between hippocampus RA concentration and memory performance in the Ymaze for the four groups. We observed that hippocampus RA level and memory were closely related for RA concentration values below 34 pmol/g, while plots over this value were distributed along a plateau (Figure 4D). This final result underlines the need for a minimal level of RA in the hippocampus to function correctly and permit memory performance.

DISCUSSION

In summary, our work showed that a vit. A enriched diet constitutes a powerful strategy for improving hippocampus neuronal network and memory performance in aged rats. In addition, we showed for the first time that memory improvement is directly related to hippocampus RA concentration increase induced by vit. A supplementation.

Retinoids availability reduces with aging

The impact of aging on retinoid signaling has been previously studied indirectly by measuring expression levels of related enzymes and receptors, in both humans and rodent models [START_REF] Azaïs-Braesco | Vitamin A status in the institutionalized elderly. Critical analysis of four evaluation criteria: dietary vitamin A intake, serum retinol, relative dose-response test (RDR) and impression cytology with transfer (ICT)[END_REF][START_REF] Enderlin | Age-related decreases in mRNA for brain nuclear receptors and target genes are reversed by retinoic acid treatment[END_REF][START_REF] Etchamendy | Vitamin A deficiency and relational memory deficit in adult mice: relationships with changes in brain retinoid signalling[END_REF][START_REF] Feart | Aging affects the retinoic acid and the triiodothyronine nuclear receptor mRNA expression in human peripheral blood mononuclear cells[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. Here, on the basis of a previously described method (Kane and Napoli, 2010), we have designed a highly sensitive analytical method with LC-ESI-MS/MS to directly measure RA levels into the hippocampus. The small quantities of tissue required (> 15mg) enable regional specificity.

Remarkably, our results revealed that levels of RA in the hippocampus of young rats are within the same range than those found in the liver. These results are in accordance with a previous report [START_REF] Kane | Quantification of endogenous retinoids[END_REF], highlighting the importance of RA levels in the hippocampus.

Here, we demonstrated that both retinol and RA concentrations were reduced in the serum of aged rats. The observation of a decreased retinol level in the serum was in accordance with previous reports [START_REF] Chevalier | Dietary restriction alters retinol and retinol-binding protein metabolism in aging rats[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF]. Additionally, we revealed that vit A dietary supplementation for 4 weeks is sufficient to significantly increase serum levels of retinol, as well as RA in aged rats. Our data showed that levels of RA in the serum and in the hippocampus were variable between rats. This variability was reliable for a same animal between the serum and the hippocampus, indicating that the variability was not due to technical aspects of RA measurement. This suggests a high inter-individual variability, however we did not find a factor explaining these differences (e.g. no correlation with weight or weight gain). Our data also highlighted the very low level of RA in the hippocampus of aged rats. Considering that RA directly activates the expression of its own receptors, this confirms previous results showing an agerelated decreased expression of RA receptors [START_REF] Enderlin | Age-related decreases in mRNA for brain nuclear receptors and target genes are reversed by retinoic acid treatment[END_REF][START_REF] Etchamendy | Alleviation of a selective age-related relational memory deficit in mice by pharmacologically induced normalization of brain retinoid signaling[END_REF].

The present study demonstrates that a reduced RA concentration in the hippocampus is an important trait in the physiopathology of aging. Considering that RA is critical for many processes in the adult brain [START_REF] Olson | Significance of vitamin A to brain function, behavior and learning[END_REF][START_REF] Shearer | A vitamin for the brain[END_REF][START_REF] Stoney | A Vitamin on the Mind: New Discoveries on Control of the Brain by Vitamin A[END_REF], maintaining sufficient RA levels through aging might constitute an important issue to prevent aged-related brain dysfunctions in humans. Indeed, since hypoactivity of retinoid signaling has also been demonstrated in the serum of aged humans [START_REF] Feart | Aging affects the retinoic acid and the triiodothyronine nuclear receptor mRNA expression in human peripheral blood mononuclear cells[END_REF], we can suppose that, as in rodents, RA concentration might be decreased in aged humans. Our experiment demonstrates that vit A. supplementation of aged rats allows restoration of hippocampus RA to the level of young rats. This level of RA seems to be well regulated when vit. A is provided to aged rats through the diet, with no risk of overloading the brain with RA, as seen with stable levels in supplemented young rats. Conversely, direct injection of RA in aged rats induced an increase of hippocampus RA level well over the one of young rats (data not shown). Dietary vit. A supplementation thus appears as a promising way to maintain RA levels through aging. However, while our data clearly demonstrate the advantages of enriching vit. A in the diet, it should be called to mind that excess of vitamin A can be toxic [START_REF] Adams | The neurobehavioral teratology of retinoids: a 50-year history[END_REF]. Therefore, toxicity studies will be needed before dietary vit. A supplementation is envisaged. Here, we found that RA concentration in the hippocampus of aged rats significantly correlates with RA but not retinol level in the serum, which suggests that part of brain RA directly comes from serum RA. This result enhances previous knowledge about sources of RA in the hippocampus (serum and local synthesis) (Arendt et al., 2015b;[START_REF] Goodman | Patterning of retinoic acid signaling and cell proliferation in the hippocampus[END_REF][START_REF] Kurlandsky | Plasma delivery of retinoic acid to tissues in the rat[END_REF][START_REF] Lane | Role of retinoid signalling in the adult brain[END_REF]. In addition, our data suggests that RA level in the serum could be relevant as a marker of brain RA levels, and might serve to identify aged people at risk of RA deficiency.

Retinoids metabolism in the liver is dysregulated with aging

In this paper, we addressed the question of the origin of retinol and RA collapse occurring with aging. We showed that in the liver of aged rats, retinol and RA were present in remarkably high concentrations compared to young animals. While it was already known that hepatic retinol stock increases with age [START_REF] Chevalier | Dietary restriction alters retinol and retinol-binding protein metabolism in aging rats[END_REF][START_REF] Pallet | Aging decreases retinoic acid and triiodothyronine nuclear expression in rat liver: exogenous retinol and retinoic acid differentially modulate this decreased expression[END_REF][START_REF] Van Der Loo | Age-related changes of vitamin A status[END_REF], increased RA level in the liver of aged rats constitutes an original result of importance. It is noteworthy that the hepatic variations of retinol and RA level with aging are diametrically opposed to their variations in the serum. This discrepancy suggests that aging induces an important dysregulation of retinol / RA release from the liver to the serum. Thus, aging leads to hepatic trapping not only of vit.A, but also of RA, which is no longer captured by the bloodstream. Importantly, dietary vit.A supplementation reverses these processes and restores circulating levels of RA.

The mechanism for RA export from the liver to the blood is unknown, as well as the nature of proteins responsible for RA transport in the blood, other than albumin [START_REF] Sani | Determination of binding affinities of retinoids to retinoic acid-binding protein and serum albumin[END_REF][START_REF] Smith | The plasma transport and metabolism of retinoic acid in the rat[END_REF]. This study does not give additional clue to understand mechanisms of RA export from the liver to the blood, and its deregulation occurring with age. However, we can postulate that age-related accumulation of retinol and RA in the liver is due to a dysregulation of several enzymes involved in retinol and RA metabolism. Indeed, retinol entry via RBPR2 transporter is increased, concomitantly with decreased metabolism, i.e. esterification and dehydrogenation through LRAT and RALDH2 enzymes respectively. In parallel, RA increase is amplified by a lack of its degradation due to CYP26A1 decreased expression (Figure 5).

Remarkably, vit. A supplementation restored the levels of mRNA expression of all of these metabolism enzymes together with RA levels in the blood and in the hippocampus. This nutritional supplementation, by increasing retinol serum levels, may regulate retinoid metabolism enzymes in the liver and thus, create a virtuous circle that may release RA to the serum, and consequently to the hippocampus. Moreover, the restoration by the supplementation of the retinoid mobilization from the liver may be explained by the bypass pathway [START_REF] Goodman | Tissue distribution and metabolism of newly absorbed vitamin A in the rat 7[END_REF][START_REF] Li | The multifaceted nature of retinoid transport and metabolism[END_REF]. This pathway processes dietary retinol without passing by the liver. A more recent study found that in neonates under vitamin A-marginal condition, supplementation with vitamin A increases the part of retinol in the brain that is coming from this bypass [START_REF] Hodges | Vitamin A Supplementation Increases the Uptake of Chylomicron Retinyl Esters into the Brain of Neonatal Rats Raised under Vitamin A-Marginal Conditions[END_REF]. Further investigations with radiolabeled retinol and metabolic models, [START_REF] Hodges | Vitamin A supplementation redirects the flow of retinyl esters from peripheral to central organs of neonatal rats raised under vitamin Amarginal conditions[END_REF][START_REF] Hodges | Vitamin A Supplementation Increases the Uptake of Chylomicron Retinyl Esters into the Brain of Neonatal Rats Raised under Vitamin A-Marginal Conditions[END_REF] are needed to determine the precursory events that initiate the dysregulation occurring with aging and how precisely dietary vit. A supplementation arrests it.

Normalization of hippocampal retinoic acid level corrects hippocampal network and agerelated memory deficits in rats

Dendritic arborization of neurons determine their capacity to adequately process in brain networks [START_REF] Jan | Branching out: mechanisms of dendritic arborization[END_REF]. Quality and complexity of dendritic branching of hippocampal neurons is thus crucial for cognitive and memory processes [START_REF] Kulkarni | The dendritic tree and brain disorders[END_REF]. Aging is characterized by a shrinkage of dendritic arborization, particularly in the hippocampus [START_REF] Bartsch | The hippocampus in aging and disease: From plasticity to vulnerability[END_REF][START_REF] Burke | Neural plasticity in the ageing brain[END_REF], which is presumably an important determinant for agerelated cognitive decline [START_REF] Bartsch | The hippocampus in aging and disease: From plasticity to vulnerability[END_REF][START_REF] Kulkarni | The dendritic tree and brain disorders[END_REF]. In our study, we focused on the CA1 region, a subfield of the hippocampus that is highly involved spatial memory as tested in the Y-maze test. Furthermore, CA1 region expresses RARα, RARγ and RXRβ [START_REF] Goodman | Patterning of retinoic acid signaling and cell proliferation in the hippocampus[END_REF][START_REF] Krezel | Differential expression of retinoid receptors in the adult mouse central nervous system[END_REF], which makes it sensitive to modulation by dietary vitamin A. We confirmed that aged rats exhibit a strong reduction of dendritic arborization, in terms of total length as well as branching density, in the CA1 region of the dorsal hippocampus. We further demonstrated that dietary vit. A supplementation totally normalized dendritic arborization of aged rats to the levels of young rats, in terms of length and branching. The mechanisms by which vit. A supplementation restores dendritic arborization in CA1 region may be multifactorial, which would need further investigation. However, we can assume that these mechanisms involve directly and/or indirectly RA, the active metabolite of vit. A in the brain. As a matter of fact, it is established that RA via its nuclear receptors is a developmental morphogen responsible for early patterning of the brain [START_REF] Maden | Retinoic acid in the development, regeneration and maintenance of the nervous system[END_REF]. At adult age, RA is also described as a key player in plasticity. Indeed, retinoic acid and its nuclear receptors (RAR, RXR) are involved in dendritic spines formation [START_REF] Chen | All-trans-retinoic acid stimulates translation and induces spine formation in hippocampal neurons through a membrane-associated RARalpha[END_REF] as well as in synaptic plasticity, notably in the hippocampus [START_REF] Aoto | Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity[END_REF][START_REF] Chen | Synaptic retinoic acid signaling and homeostatic synaptic plasticity[END_REF][START_REF] Chiang | An essential role for retinoid receptors RARbeta and RXRgamma in long-term potentiation and depression[END_REF]. In accordance with this, we previously showed that RA in aged rodents improves synaptic plasticity, i.e. long term potentiation and expression of genes involved in synaptic plasticity [START_REF] Etchamendy | Alleviation of a selective age-related relational memory deficit in mice by pharmacologically induced normalization of brain retinoid signaling[END_REF][START_REF] Féart | Differential effect of retinoic acid and triiodothyronine on the age-related hypo-expression of neurogranin in rat[END_REF]. RA also facilitates neuronal survival and neurite outgrowth and induces neuronal differentiation [START_REF] Chen | All-trans-retinoic acid stimulates translation and induces spine formation in hippocampal neurons through a membrane-associated RARalpha[END_REF][START_REF] Christie | Retinoid supplementation of differentiating human neural progenitors and embryonic stem cells leads to enhanced neurogenesis in vitro[END_REF][START_REF] Takahashi | Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures[END_REF]. At the molecular level, RA binds to nuclear receptors which modulates gene transcription. Thus, future investigation with transcriptomic approaches may identify all target genes of RA in relation to plasticity of dendritic arborization through aging.

Finally, our study reveals that restoration of dendritic arborization and RA level in the hippocampus by dietary vit. A supplementation is concomitant with a reinstatement of memory performance in aged rats. The direct role of hippocampal RA in the modulation of memory performances along the life has already been argued [START_REF] Etchamendy | Vitamin A deficiency and relational memory deficit in adult mice: relationships with changes in brain retinoid signalling[END_REF][START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF] but without evidence for a direct link. Our present study reveals a non-linear correlation between RA levels in the hippocampus and memory performance of aged rats. Importantly, our results are in accordance with a recent study showing a relation between blood levels of retinol and age-related cognitive decline in humans [START_REF] Huang | Diminished circulating retinol and elevated α-TOH/retinol ratio predict an increased risk of cognitive decline in aging Chinese adults, especially in subjects with ApoE2 or ApoE4 genotype[END_REF]. In the present work, we assessed episodic-like memory since it is particularly sensitive to aging processes. In non-supplemented aged rats, the correlation between memory performance and RA levels in the hippocampus was highly significant, with the best performing rats exhibiting the highest levels of RA in the hippocampus. Interestingly, in supplemented aged rats, the correlation becomes low (no significant), similarly to young rats. This reflects high memory performance enabled by a sufficient amount of hippocampal RA. The non-linear fit of the data reveals that most of rats with high memory performance (at the level of young rats) exhibit a concentration of RA on the hippocampus higher than 34 pmol/g. This threshold might represent the minimal and necessary concentration of RA in the rat hippocampus for the elaboration of neuronal enriched circuits, required for optimal memory processes.

In conclusion, these original results underpinned the importance of RA signaling for hippocampus functionality. This brings new knowledge on the mechanisms linking alteration of vit. A metabolism, RA signaling in the brain and aged-related cognitive impairments. Figure 5: Recapitulative illustration. RA is synthetized in the liver and circulate through the blood stream to access the hippocampus. Hippocampal RA is necessary for neuronal circuits that enable memory performance.

  p=0.0064. (D) Plot of RA level in the hippocampus as a function of RA level in the serum for aged rats, supplemented or not with dietary vit. A. Histogram are represented as mean ± SEM with individual data points. Young rats: white bars; young + vit. A supplementation: light orange bars; aged rats: grey bars; aged rats + vit. A supplementation: dark orange bars. a-c: values significantly different.

Figure 2 :

 2 Figure 2: Analysis of retinol and RA metabolism in the liver. The illustration (top right) draws the involvement of RBPR2, LRAT, REH, RALDH2 and CYP26A1 proteins in retinol metabolism in the liver. atRA: all-trans retinoic acid. (A) levels of retinol (µmol/g) in the liver for young and aged rats, supplemented or not with dietary vit. A (n=8 per group). Two-way ANOVA: Age effect, p<0.0001;

Figure 3 :

 3 Figure 3: Dendritic morphology of pyramidal neurons in the CA region of the dorsal hippocampus. (A) Representative neurons from young rat (first left), young rat supplemented (second left), aged rat (third left) and aged rat supplemented (right), following Golgi staining and Imaris 3D reconstruction. Apical pole: young, n=24; young + vit. A, n=21; aged, n=20; aged + vit.A, n=20. Basal pole: young, n=20; young + vit. A, n=21; aged, n=20; aged + vit.A, n=20. (B, C) Total length (µm) of dendritic trees for the apical (B) and basal (C) poles of CA1 pyramidal neurons. Neurons from (A) are represented with blue diamond. (B) Apical pole: Two-way ANOVA: Age effect, p=0.0246; vit. A effect: p=0.1375; interaction: p=0.00. Bonferroni post-hoc comparison with the group young rats: young + vit. A p=0.5933; aged p=0.0004; aged +vit. A p>0.9999. (C) Basal pole: Two-way ANOVA: Age effect, p=0.0989; vit. A effect: p=0.0407; interaction: p=0.0003. Bonferroni post-hoc comparison with the group young rats: young + vit. A p=0.6869; aged p=0.0008; aged +vit. A p>0.9999. (D, E) Dendritic length of apical (D) and basal (E) poles of CA1 neurons classified by dendrite level. (D) Apical pole: two-way ANOVA: group effect, p=0.0012; dendritic level effect: p<0.0001; interaction: p=0.7527. (E) Basal pole: two-way ANOVA: group effect, p=0.0005; dendritic level effect: p<0.0001; interaction: p=0.0169. Bonferroni post-hoc comparison between young and aged rats significant for levels 3 (p=0.0054), 4 (p=0.0003) and 5 (p=0.0159). (F, G) Number (#) of branches for each apical and basal poles of CA1 neurons in the 4

Figure 4 :

 4 Figure 4: Spatial memory performance of aged rats match with hippocampal RA levels. (A, B) Total distance and time spent in individuals Y-maze arms for the 4 groups: young, n=9; young + vit. A, n=10; aged, n=10; aged+ vit. A, n=10. (A) Total distance (cm) in Y-maze test. Two-way ANOVA, Age effect: p=0.5666, vit. A effect: p=0.2347, interaction: p=0.4901. (B) Absolute time (s) spent in start (empty bar), familiar (dots pattern) and new arms (square pattern) of the Y-maze. Separate analysis for each group with one-way ANOVA. Young: p=0.0052; familiar vs. new arm: p=0.0004. Young + vit. A: p<0.0001; familiar vs. new arm: p<0.0001; start vs new arm: p=0.0011. Aged: p=0.0844; Aged + vit.A: p=0.0223; familiar vs. new arm: p=0.1556; start vs new arm: p=0.0279. (C) Proportion (%) of time spent in the new arm, comparatively to the familiar arm (chance level = 50 %). Two-way ANOVA: Age effect: p<0.0001; vit. A effect: p=0.0007; interaction: p=0.2215. (D) Plot of percent time spent in the new arm of the Y-maze (memory performance) as a factor of RA levels in the hippocampus. Dashed horizontal line at 50 % represents chance level (absence of memorization). Light-orange straight line represents linear regression for young + young+vit. A rats. Grey straight line represents linear regression for aged rats. Orange straight line represents linear regression for aged rats supplemented with dietary vit. A. Curved black line represents nonlinear regression of aged rats, supplemented and not supplemented (equation: y=(85.3±5.3*x)/(17.8±5.1 + x), goodness of fit: r² = 0.514). Histogram are represented as mean ± SEM with individual data points. Young rats: white bars; young + vit. A supplementation: light

1: RA and retinol levels in young and aged rats, supplemented or not with dietary vit. A.

	(A) levels of retinol (µM) in the serum for young and aged rats, supplemented or not with dietary
	vit. A (n=8 per group). Two-way ANOVA: Age effect, p<0.0001; vit. A effect: p=0.0005; interaction:
	p=0.0015; Bonferroni posthoc comparison with the group young rats: young + vit. A p>0.9999;
	aged p<0.0001; aged + vit. A p<0.0001; comparison between aged and aged + vit. A, p<0.0001.
	Bonferroni posthoc comparison between aged and aged + vit. A groups: p<0.0001. (B) levels of RA
	(pmol/ml) in the serum for young and aged rats, supplemented or not with dietary vit. A (n=10 per
	group except for aged rats, n=9). Two-way ANOVA: Age effect, p=0.1555; vit. A effect: p=0.0484;
	interaction: p=0.0086; Bonferroni posthoc comparison with the group young rats: young + vit. A
	p>0.9999; aged p=0.0335; aged +vit. A p>0.9999 comparison between aged and aged + vit. A:
	p=0.0111. (C) Levels of RA (pmol/g) in the hippocampus (HPC) for the 4 groups (n=10 per group
	except for young rats, n=9). Two-way ANOVA: Age effect, p=0.0056; vit. A effect: p=0.0074;
	interaction: p=0.0398. Bonferroni post-hoc comparison with the group young rats: young + vit. A
	p>0.9999; aged p=0.0067; aged +vit. A p>0.9999. Comparison between aged and aged + vit. A:

ACKNOWLEDGMENTS

The authors would like to thank staff from NutriNeuro animal facility and office. The microscopy was done in the Bordeaux Imaging Center a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging supported by the French

FUNDING

This work was supported by Institut CARNOT LISA (ANR) and the Région Nouvelle Aquitaine. Mass spectrometry equipment was supported by the Région Nouvelle Aquitaine and the Platform Proteome (https://proteome.cgfb.u-bordeaux.fr).

COMPETING INTEREST

No competing interest is declared by the authors.

National Research Agency (ANR-10-INBS-04). The help of Christel Poujol and Sébastien Marais is acknowledged.

AUTHORS CONTRIBUTION

Conceptualization


Hélène Licandro

Phu Ha Ho

Thi Kim

Chi Nguyen

Awanwee Petchkongkaew

Hai Van Nguyen

Son Chu-Ky

Thi Viet

Anh Nguyen

Da Lorn

Yves Waché

How fermentation by lactic acid bacteria can address safety issues in legumes food products?

Keywords: legumes, lactic acid bacteria, phytates, biogenic amines, mycotoxins, food safety

Fermented Asian foods have recently gained popularity, crossing from Asian communities to mainstream markets, in many western countries. However, less attention has been paid to the safety of these foods. In South-East Asia fermented products are still produced following traditional methods. Therefore, consumers are not confident in their microbial safety. The challenges awaiting fermentation in South-East Asia are thus to improve safety and quality in a sustainable system producing tasty and typical fermented products. A possible solution could be the use of starter cultures able to increase the safety of food stuffs. Starters could also help to standardize the production process and reduce ripening times. The current review addresses the role of lactic acid bacteria on the microbiological and chemical safety of Asian legume based fermented products. In particular, their role in the reduction of anti nutritional compounds (e.g. phytates) and protein allergenicity is discussed. Moreover, starters can inihibit the development of amino acid decarboxylating microbes preventing the accumulation of biogenic amines, they can also be useful to reduce the accumulation of mycotoxins and inhibit pathogens' development. Finally, their role in the degradation of pesticides is analyzed.

Introduction

The food system is facing a crisis as consumers are confused by ambivalent information arising all along the food chain. Plant products bring on the one hand vitamins and micronutrients, they have a positive impact on the environment but on the other hand, they might contain non-neglectable amounts of antinutritional factors (ANFs), toxic chemicals and toxins. For their culture, agroindustry promotes the facilitated culture of GMO while scientific studies reveal that the use of pesticide-resisting varieties results in many risks, including the increase in the level of pesticide in culture, in the soil and, ultimately, in the food [START_REF] Bonny | Genetically Modified Herbicide-Tolerant Crops, Weeds, and Herbicides: Overview and Impact[END_REF][START_REF] Tsatsakis | Impact on environment, ecosystem, diversity and health from culturing and using GMOs as feed and food[END_REF]). Some of these issues might find solutions in the future by the selection of low ANF-varieties, by improving cultural techniques, decreasing the use of chemicals, developing storage techniques avoiding mold contamination etc. but this will take time, especially in South-East Asia where farmers are often not enough trained and have economic limitations to implement the required changes in culture practices. Microorganisms functionalities in fermented foods can bring several advantages to consumers [START_REF] Tamang | Functional properties of microorganisms in fermented foods[END_REF]. As an efficient, rapid and low cost solution to nutritional and food safety concerns, the use of bio-remediation starters can be added to these benefits and this will be discussed in the present work with a special focus on legumes.

Fermented leguminous in South-East Asia

Legumes such as soybeans, black beans, green beans, peanuts... are common ingredients in South-East Asia for food production. They are popular for various reasons. In one hand, when grown, they have the possibility to supply nitrogen to the soil through a mycorrhizal association that makes this family of plant an actor of rotations in organic agriculture. On the other hand, their protein content is also very high making them good nutritional candidates to replace meat or milk, which production is an important source of greenhouse gas, and a big consumer of plant and water resources especially in developing countries [START_REF] Tubiello | Greenhouse gas emissions due to agriculture[END_REF]. In particular, soybeans are most used for the production of traditional fermentation products such as fermented soybean paste (Ban Tuong, Cu Da Tuong -Vn, Nam Dan Tuong -Vn, Sieng -Kh), fermented soybean curd (Chao -Vn), Tauxi -Ha Giang Dau Xi -Vn), alkaline fermentation of soybeans (natto-like) (Thua Nao -Th). Fermentation of these beans can have an important nutrient and health impact [START_REF] Cao | Bioactivity of soy-based fermented foods: A review[END_REF][START_REF] Jayachandran | An insight into the health benefits of fermented soy products[END_REF].

However, production of traditional fermentation products in South-East Asia is now managed by experience, produced mainly by manual households, with rudimentary production equipment, mostly fermented by endogenous microorganisms. Raw materials as well as technologies have not been standardized. These products are characterized by the local cultural identity. Despite their important sensorial role in Asian food, bringing for instance the umami taste to the meals [START_REF] Hajeb | Umami taste components and their sources in Asian foods[END_REF], production management and quality measurement are needed to improve quality and minimize food safety hazards. Food safety issues in traditional fermented food from South-East Asia have been reviewed recently [START_REF] Anal | Food safety risks in traditional fermented food from South-East Asia[END_REF] and soybeans, as a protein-rich matrice, are exposed to a biogenic amine risk [START_REF] Park | Occurrence and reduction of biogenic amines in traditional Asian fermented soybean foods: A review[END_REF].

Lactic acid bacteria (LAB) are usually not considered as the most important bacteria in traditional fermentation of legumes although they have an important role and are numerous in the vietnamese tuong and in the cambodian sieng.

Antinutritional factors: can metabolic properties of LAB respond the challenges?

Anti-nutritional factors (ANFs) are one of the main limitations for the development of legume-based foods. Since the early 80's, spontaneous fermentation, generally household, has been reported to degrade some leguminous ANFs such as phytates, trypsin inhibitors and lectins (Chompreeda & Field, 1984a;Reddy & Pierson, 1994). Most of the analyzed fermented soy derivatives contain very small amount of trypsin inhibitors and isoflavones, compared to the raw soybean, but still contain phytates [START_REF] Anderson | Compositional changes in trypsin inhibitors, phytic acid, saponins and isoflavones related to soybean processing[END_REF]. However, it was not clear if microorganisms, including lactic acid bacteria (LAB) and yeasts, played an important role and even less what was their way of action. Hence, controlled lactofermentations were carried out. For instance, [START_REF] Sindhu | Probiotic fermentation of indigenous food mixture: effect on antinutrients and digestibility of starch and protein[END_REF] demonstrated that LAB (Lactobacillus casei and Lactobacillus plantarum) can reduce phytates, polyphenols and trypsin inhibitors in a food mixture containing green grams, with a higher effect when inoculated after a first fermentation with yeasts (Saccharomyces boulardii). However, LAB did not seem to have any effect on agglutinins (also called lectins) [START_REF] Ayyagari | Lectins, trypsin inhibitors, BOAA and tannins in legumes and cereals and the effects of processing[END_REF][START_REF] Barkholt | Protein modification by fermentation: effect of fermentation on the potential allergenicity of pea[END_REF]Motarjemi & Nout, 1996). Accurate characterizations of LAB activities are underway with the objective to develop appropriate starters and processes, particularly for tannins and phytates removing.

Metabolism of tannins or other polyphenols by LAB has been characterized only in a few plant fermentations including tempeh [START_REF] Starzyńska-Janiszewska | Effect of controlled lactic acid fermentation on selected bioactive and nutritional parameters of tempeh obtained from unhulled common bean (Phaseolus vulgaris) seeds[END_REF] and sorghum [START_REF] Svensson | Phenolic acids and flavonoids in nonfermented and fermented red sorghum (Sorghum bicolor (L.) Moench)[END_REF]. Among LAB, L. plantarum, Lactobacillus paraplantarum, Lactobacillus pentosus seem to be the only species capable of degrading hydrolysable tannins through a tannase activity (tannin acyl hydrolase, EC 3.1.1.20) which hydrolyzes ester bounds of tannic acid, thus releasing glucose and gallic acid (Osawa et al., 2000;[START_REF] Vaquero | Tannase activity by lactic acid bacteria isolated from grape must and wine[END_REF]Rodriguez et al., 2009). Most of tannase producers were found in fermented vegetables but also in human feces. In L. plantarum, tannase is very well characterized. Its activity was demonstrated and characterized by [START_REF] Rodríguez | Characterization of tannase activity in cell-free extracts of Lactobacillus plantarum CECT 748 T[END_REF] and genetic analysis showed it constitutes a novel family of tannases [START_REF] Iwamoto | Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917 T[END_REF]. LAB tannases are intracellular.

A L. plantarum strain has been reported to produce a very efficient tannase during anaerobic fermentation [START_REF] Aguilar-Zárate | Gallic acid production under anaerobic submerged fermentation by two bacilli strains[END_REF]. Genes involved in tannins degradation are regulated in a coordinated way and are inducible by tannin and other phenolic compounds (Reveron et al., 2017). Characterization of fermented cassava LAB allowed identifying uncommon tannase producers such as Weissella cibaria and Leuconostoc mesenteroides ssp.

mesenteroides [START_REF] Kostinek | Characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures[END_REF].

Degradation of phytates during food process is very inconstant, depending on the process (soaking, heating, milling …) and on the plant species [START_REF] Gibson | Improving the bioavailability of nutrients in plant foods at the household level[END_REF]. Soaking and germination can activate plant endogenous phytases in most cereals and legumes [START_REF] Holzapfel | Use of starter cultures in fermentation on a household scale[END_REF]. The role of endogenous plant phytases during fermentation is still under debate. For some authors, phytate reduction is rather the consequence of plant phytases activation during fermentation than of microbial phytases [START_REF] Reale | The importance of lactic acid bacteria for phytate degradation during cereal dough fermentation[END_REF][START_REF] Shirai | Ability of some strains of lactic acid bacteria to degrade phytic acid[END_REF]. Indeed, LAB decrease and thus optimize the pH for plant phytases that are more active between pH 5 and pH 4 [START_REF] Gänzle | Enzymatic and bacterial conversions during sourdough fermentation[END_REF]. Moreover, production of organic acids by LAB could play a synergic effect by complexing with minerals from phytates, thus making them more accessible for phytases [START_REF] Maenz | The effect of minerals and mineral chelators on the formation of phytase-resistant and phytase-susceptible forms of phytic acid in solution and in a slurry of canola meal[END_REF]. On the contrary, other authors claim that phytase activity is very variable depending on the plant species, thus plant phytase activities would not be sufficient in most fermentations [START_REF] Cossa | Variabilities of total and phytate phosphorus contents as well as phytase activity in wheat[END_REF].

Phytases have been found in yeasts, fungi and bacteria. Generally, they are intracellular.

Bacterial phytases have been detected in various ubiquitous genera such as Bacillus, Pseudomonas, Klebsiella, Enterobacter, Escherichia and in anaerobic ruminal bacteria [START_REF] Konietzny | Molecular and catalytic properties of phytate-degrading enzymes (phytases)[END_REF]. A few LAB strains have been shown to degrade, at least partially, phytates. The strains belonged to Pediococcus pentosaseus in millet and soya [START_REF] Raghavendra | Phytate-degrading Pediococcus pentosaceus CFR R123 for application in functional foods[END_REF], Leuconostoc mesenteroides, L. plantarum, Lactobacillus acidophilus and P. pentosaseus in wheat [START_REF] Lopez | Strains of lactic acid bacteria isolated from sour doughs degrade phytic acid and improve calcium and magnesium solubility from whole wheat flour[END_REF][START_REF] Cizeikiene | Phytase activity of lactic acid bacteria and their impact on the solubility of minerals from wholemeal wheat bread[END_REF], some Lactobacillus species in soy milk [START_REF] Tang | Phytase activity from Lactobacillus spp. in calcium-fortified soymilk[END_REF], L. acidophilus, Lactobacillus buchneri and L. plantarum in lupin [START_REF] Camacho | Nutritional quality of lupine (Lupinus albus cv. Multolupa) as affected by lactic acid fermentation[END_REF][START_REF] Fritsch | Fermentation performance of lactic acid bacteria in different lupin substrates-influence and degradation ability of antinutritives and secondary plant metabolites[END_REF], Streptococcus thermophilus and Bifidobacterium infantis in co-fermentation in soymilk [START_REF] Lai | Effect of lactic fermentation on the total phenolic, saponin and phytic acid contents as well as anti-colon cancer cell proliferation activity of soymilk[END_REF].

A few studies aimed to screen phytase activities among LAB strains. [START_REF] Anastasio | Selection and use of phytate-degrading LAB to improve cereal-based products by mineral solubilization during dough fermentation[END_REF] have found 29 phytase-producers among 150 LAB, most of which isolated from wheat fermentation, whereas Songré-Ouattara et al. ( 2008) have found 30 among 155 isolated from millet fermented gruel. According to these two works, one can expect about 20% of phytases producers among LAB isolated from plant fermentation, with the highest activities for some L. plantarum, Enterococcus faecium and Lactobacillus fermentum strains. These studies constitute preliminary reports that have to be strengthened. Indeed, another study has highlighted the importance of the substrate used for the screening since on the 40 tested LAB strains able to degrade calcium phytates, only two strains (Pediococcus pentosaceus) can degrade sodium phytates [START_REF] Raghavendra | Screening, selection and characterization of phytic acid degrading lactic acid bacteria from chicken intestine[END_REF]. In addition, [START_REF] Fredrikson | Phytate degradation by micro-organisms in synthetic media and pea flour[END_REF] warned about indirect methods to detect phytates and media composition for the screening.

They did not observe reliable results for LAB. It is likely that some LAB species are considered as non-or low-phytase producers because the screening conditions do not permit phytase production in bacteria [START_REF] Konietzny | Molecular and catalytic properties of phytate-degrading enzymes (phytases)[END_REF]. Some studies have been carried out with tighter methods. [START_REF] Sreeramulu | Lactobacillus amylovorus as a phytase producer in submerged culture[END_REF] have reported that Lactobacillus amylovorus was a good producer of extracellular phytases and that extracellular activity was also detected for L. acidophilus, L. plantarum, Lactobacillus delbrueckii and L. casei. They did not detect any production with Lactococcus lactis or Lactobacillus helveticus. Moreover, [START_REF] Zamudio | Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase[END_REF] have reported the ability of six strains of LAB (P. pentosaceus, Leuconostoc mesenteroides, L. casei, L. fermentum, L. delbrueckii and L. plantarum) to partially degrade phytate (from 6 to 11%). Whereas no intracellular and low extracellular phytase activities were measured, they proved that phytate degradation was due to a non-specific acid phosphatase. [START_REF] Palacios | Characterization of an acid phosphatase from Lactobacillus pentosus: regulation and biochemical properties[END_REF] have come to the same conclusion by characterizing a L. pentosus non-specific acid phosphatase (EC 3.1.3.2) which was able to hydrolyze phytates. De [START_REF] De Angelis | Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1[END_REF] have gone further by characterizing an intracellular phytase of L. sanfranciscensis at the protein level. They did not identify the corresponding gene. This strain decreased phytate content of almost 75% in sourdough after an 8 h-incubation.

Several groups have identified and characterized efficient phytases in the Bifidobacterium genera (Haros et al., 2007;[START_REF] Tamayo-Ramos | Novel phytases from Bifidobacterium pseudocatenulatum ATCC 27919 and Bifidobacterium longum subsp. infantis ATCC 15697[END_REF][START_REF] García-Mantrana | Myo-inositol hexakisphosphate degradation by Bifidobacterium pseudocatenulatum ATCC 27919 improves mineral availability of high fibre rye-wheat sour bread[END_REF] but this genus does not fit with legumes fermentation mainly because it is strict anaerobic and fastidious. To overcome this limitation, García-Mantrana et al. ( 2016) succeeded in expressing active bifidobacterial phytases in L. casei. Nevertheless, in sourdough, the bacterial activity was low, compared to the activated plant phytases.

Actually, identifying new LAB phytase activities based on phenotypical screening seems hazardous. Otherwise, in silico analysis of LAB genomes based on known bacterial phytases (Bacillus, Escherichia spp) does not predict any phytase (personal data). Even bifidobacterial phytases found by [START_REF] García-Mantrana | Myo-inositol hexakisphosphate degradation by Bifidobacterium pseudocatenulatum ATCC 27919 improves mineral availability of high fibre rye-wheat sour bread[END_REF] are very specific. Thus, molecular screening is not feasible for LAB. However, characterizing enzymatic activities and identifying the corresponding genes in LAB strains already highlighted would be very informative to know if specific or non-specific phytases exist in some LAB strains and if these phytases would be efficient in reducing the phytate content in fermented legumes.

Decrease of protein allergenicity through LAB-mediated hydrolysis

The main legume allergens are the proteins. These complex structures are difficult to degrade.

The selection of legumes natural variants or the use of specific biotechnological processes can eliminate these particular proteins. However, they might also induce some side effects such as an increase in the protein synthesis pathways of the seed and the synthesis of other proteins that might be allergenic as well (Nowak-Wegrzyn & Fiocchi, 2009 ;Mils et al., 2009 ;[START_REF] Kroghsbo | Assessment of the sensitizing potential of processed peanut proteins in Brown Norway rats: roasting does not enhance allergenicity[END_REF][START_REF] Rahaman | Effect of processing on conformational changes of food proteins related to allergenicity[END_REF].

Plant proteins exhibits low digestibility compared to animal proteins. Poor protein digestibility can cause gastrointestinal disorders that may result in fecal excretion of proteins.

Therefore, increasing protein digestibility could reduce the levels of undigested proteins which can potentially cause food allergies due to poor absorption in the gut [START_REF] Untersmayr | The role of protein digestibility and antacids on food allergy outcomes[END_REF]. Several studies have showed that LAB fermentation increases the digestibility of plant proteins (El-Haget al., 2002;[START_REF] Pranoto | Effect of natural and Lactobacillus plantarum fermentation on in vitro protein and starch digestibilities of sorghum flours[END_REF].

For example, during fermentation of lupin and soybean, protein digestibility is improved by using LAB cultures such as Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-07 and P. pentosaceus KTU05-8. This improvement is due to the role of microbial proteolytic enzymes. These enzymes partially hydrolyze these proteins into peptides and free amino acids. These simple and soluble products are easier to digest than the whole protein (Bartkiene et al., 2013a;[START_REF] Bartkiene | Influence of the addition of lupin sourdough with different lactobacilli on dough properties and bread quality[END_REF][START_REF] Aguirre | Proteolytic activity of Lactobacillus strains on soybean proteins[END_REF]. Fermentation of soybean meal with L. plantarum or Bifidobacterium lactis showed a significant increase in the total amino acids and a low immunoreactivity. Thus, the use of fermentation to reduce or eliminate allergenicity of soy products represents an interesting opportunity to produce hypoallergenic food products from legumes (Song et al., 2008a;Song et al., 2008b).

Lactic acid bacteria as biopreservation agent against pathogenic bacteria

Besides decreasing antinutritional factors (ANFs) and allergy, lactic acid bacteria can fulfill a task of biopreservation. This word can be defined as the extension of shelf-life and food safety by the use of natural or controlled microbiota and/or their antimicrobial compounds [START_REF] Ananou | Bactericidal synergism through enterocin AS-48 and chemical preservatives against Staphylococcus aureus[END_REF]).

Fermentation by lactic acid bacteria (LAB) is one of the most common methods of food biopreservation. In this process, organoleptic properties (unique flavor, aroma compounds) of products are also developed and improved. This is an important advantage of fermentation compared to non-fermented products that have to be biopreserved without any organoleptic impact.

In South-East Asia, biopreservation strategies have been designed to avoid pathogens contamination in various products, although fermented meat as well as vegetables other than legumes, two classes being often considered as the riskiest products. For instance, [START_REF] Phan | Diversity of lactic acid bacteria in vegetable-based and meat-based fermented foods produced in the central region of Vietnam[END_REF]) determined the diversity of LAB in 25 samples of meat and vegetable fermented products in Vietnam including dua gia (bean sprouts), dua cai (cabbage), mang chua (bamboo shoots), nem chua (uncooked pork) and tre (cooked pork). The results showed that the major species found in vegetable-based products (L. plantarum, L. fermentum, and L. helveticus) were different from those identified in meat-based products (P. pentosaceus, Weissella cibaria, and Lactococcus lactis) [START_REF] Phan | Diversity of lactic acid bacteria in vegetable-based and meat-based fermented foods produced in the central region of Vietnam[END_REF]. The LAB ranged approximately from 10 6 to 10 9 CFU/g, depending on the food items [START_REF] Phan | Diversity of lactic acid bacteria in vegetable-based and meat-based fermented foods produced in the central region of Vietnam[END_REF]).

In the case of legumes, it is important to use bacteria that can develop easily on these plant matrices rich in proteins but also to take into account the specificities of contaminations. From the field, legumes are likely to bring with them sporulating bacteria. Later, fungi can develop and produce mycotoxins. Finally, depending on the handling of the products, pathogens can occur through cross-contamination from meat or vegetables. The range of possible contaminants is thus rather wide. The inhibitory mechanisms of biopreservation by LAB against spoilage organisms are destabilization of cell membrane and subsequent interference with the proton gradient, inhibition enzyme activity and creation of reactive oxygen species.

LAB strains are able to produce various antimicrobial substances such as low-molecular weight metabolites (reuterin, reutericyclin, diacetyl, fatty acids), hydrogen peroxide, antifungal compounds (propionate, phenyl-lactate, hydroxyphenyl-lactate and 3-hydroxy fatty acids), and bacteriocins that may be exploited in the biopreservation of foods [START_REF] Siedler | Bioprotective mechanisms of lactic acid bacteria against fungal spoilage of food[END_REF].

There is a wide number of bacteriocins produced by LAB and they can be classified into three From these two collections, biopreservatives LAB could be investigated for the specific problems of legume fermented products.

Biogenic amines, fermentation may be the risk, but also the solution

Microbial fermentation has been employed for millennia by mankind to prolong shelf life and improve texture and flavour of food stuffs. However, protein-rich raw materials can be a source of aminoacids which can be converted into biogenic amines (BAs) by microorganisms. Biogenic amines are nitrogenous compounds of low molecular weight occurring in several fermented foods. The main BAs present in foods are histamine, tyramine, tryptamine, putrescine, cadaverine, and phenylethylamine [START_REF] Gardini | Technological factors affecting biogenic amine content in foods: a review[END_REF]. Their accumulation is mainly due to microbial decarboxylation of aminoacids through the action of decarboxylases which remove the carboxyl group with the formation of the corresponding amine and CO2, or through the amination and transamination of aldehydes and ketones [START_REF] Alvarez | The problem of biogenic amines in fermented foods and the use of potential biogenic amine-degrading microorganisms as a solution[END_REF].

These compounds can cause toxic effects in consumers with several symptoms such as nausea, flushes, heart palpitation, headache, red rash, oral burning, and hypo-or hypertension which are more severe in consumers with an inefficient detoxification system and depend on the amount and type of BA ingested [START_REF] Ladero | Toxicological effects of dietary biogenic amines[END_REF]. Biogenic amines are produced not only by Gram-positive and Gram-negative bacteria, but also by yeasts and molds [START_REF] Gardini | Technological factors affecting biogenic amine content in foods: a review[END_REF]. However, lactic acid bacteria (LAB) are considered as the main BA producers in fermented foods and Enterococcus, Lactobacillus, Streptococcus, Lactococcus Oenococcus, Pediococcus, Weissella, Carnobacterium, Tetragenococcus, Leuconostoc, Sporolactobacillus are the main genera showing this trait (Barbieri et al., 2019). BAs production is a strain specific feature and some studies revealed that the involved enzyme is encoded by unstable plasmids [START_REF] Lucas | Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006[END_REF][START_REF] Satomi | Analysis of a 30 kbp plasmid encoding histidine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce[END_REF]. Therefore, horizontal gene transfer is essential to disseminate this ability in LAB [START_REF] Lucas | Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006[END_REF][START_REF] Satomi | Analysis of a 30 kbp plasmid encoding histidine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce[END_REF].

Although the factors influencing BAs production (e.g. temperature, salt concentration, and pH) are well known, it is difficult to prevent their accumulation in fermented foods, since the fermentation conditions cannot be easily modified and the aminobiogenic ability is straindependent. Several strategies have been proposed to prevent BAs accumulation in foods; however, they can decrease the nutritional value and the organoleptic features of foods (for a review see Naila et al., 2010). In the case of legumes, the boiling allows the elimination of BAs, unfortunately, for sprouted legumes, this approach permits only a reduction of their content [START_REF] Shalaby | Changes in biogenic amines in mature and germinating legume seeds and their behavior during cooking[END_REF]. One of the most promising strategies is the use of amine oxidizing starter cultures. Three different classes of oxidases have been described: flavin-containing monoamine oxidases (FlavAO), copper containing amine oxidases (CuAO), and multi-copper oxidases (MCO). They catalyze the oxidative deamination of BAs forming the corresponding aldehydes, hydrogen peroxide and ammonia [START_REF] Guarcello | Selection of amine-oxidizing dairy lactic acid bacteria and identification of the enzyme and gene involved in the decrease of biogenic amines[END_REF]. [START_REF] Kim | Isolation of biogenic aminesdegrading strains of Bacillus subtilis and Bacillus amyloliquefaciens from traditionally fermented soybean products[END_REF] isolated strains of Bacillus subtilis and Bacillus amyloliquefaciens from fermented soybean foods. They observed the ability of B. subtilis to degrade putrescine and cadaverine and of B. amyloliquefaciens to oxidize histamine and tyramine. Similarly, [START_REF] Kang | Potential for application as a starter culture of tyramine-reducing strain[END_REF] showed the ability of B. subtilis and B. amyloliquefaciens strains to reduce tyramine in Cheonggukjang. [START_REF] Eom | Biogenic amine degradation by Bacillus species isolated from traditional fermented soybean food and detection of decarboxylase-related genes[END_REF] isolated from buckwheat sokseongjang, a Korean traditional fermented soybean food, 3 strains (B. subtilis HJ0-6, B. subtilis D'J53-4, and B. idriensis RD13-10) which were not only able to degrade histamine and tyramine but also unable to produce them. Finally, [START_REF] Lee | Reduction of biogenic amines during miso fermentation by Lactobacillus plantarum as a starter culture[END_REF] proposed the use of L. plantarum strains to reduce BAs content during Miso fermentation.

The possibility to select new amine oxidizing starter cultures could represent a promising strategy to reduce BAs content in legumes based fermented foods where traditional methods are inadequate or it is necessary to eliminate BAs already formed during the fermentation process.

Mycotoxins in leguminous: can fermentation degrade what fungi have made?

Fermentation is the chemical process which converts substrates into product(s) by using microorganisms for example fungi, yeast and bacteria. There are many undesirable substances contaminating foods and feeds that are harmful to human and animal health. These include mycotoxins, which are widely present in food and feeds commodities. Microorganisms including fungi, yeast, and bacteria are able to eliminate mycotoxins. Additionally, various studies have been extensively reporting about their mycotoxin degradation mechanisms such as cell wall-binding, enzyme degrading or structure modification. However, these mechanisms of actions depend on strains of microorganisms. [START_REF] El-Sharkawy | Microbial transformation of zearalenone: Reduction, hydroxylation, and methylation products[END_REF][START_REF] Kakeya | Biotransformation of the mycotoxin, zearalenone, to a non-estrogenic compound by a fungal strain of Clonostachys sp[END_REF][START_REF] Kakeya | Biotransformation of the mycotoxin, zearalenone, to a non-estrogenic compound by a fungal strain of Clonostachys sp[END_REF][START_REF] Vekiru | Cleavage of zearalenone by Trichosporon mycotoxinivorans to a novel nonestrogenic metabolite[END_REF][START_REF] El-Nezami | Binding Rather Than Metabolism May Explain the interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative α-Zearalenol[END_REF][START_REF] Zhao | Cross-linked chitosan polymers as generic adsorbents for simultaneous adsorption of multiple mycotoxins[END_REF]. Lactic acid bacteria (LAB) can be extensively found in various fermented foods (meat, vegetable, and milk). They are widely used to ferment and preserve foods and feeds in place of chemical preservatives due to their lower cost and better safety.

They can produce antimicrobial and antagonistic compounds for the control of pathogenic bacteria/fungi and spoilage microflora. Additionally, the LAB are widely applied to reduce mycotoxins. The capability of LAB to remove mycotoxin has been reported more than two decades ago for several toxins such as aflatoxin B1 [START_REF] Haskard | Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG[END_REF][START_REF] El-Nezami | Binding Rather Than Metabolism May Explain the interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative α-Zearalenol[END_REF][START_REF] Lahtinen | Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG[END_REF][START_REF] Taheur | Adsorption of aflatoxin B1, zearalenone and ochratoxin A by microorganisms isolated from Kefir grains[END_REF], zearalenone [START_REF] El-Nezami | Chemical moieties and interactions involved in the binding of zearalenone to the surface of Lactobacillus rhamnosus strains GG[END_REF][START_REF] Adunphatcharaphon | In Vitro Screening of Potential Plant-Derived Lactic Acid Bacteria for Zearalenone Removal[END_REF][START_REF] Long | Removal of zearalenone by strains of Lactobacillus sp. isolated from rumen in vitro[END_REF], ochratoxin A [START_REF] Fuchs | Detoxification of patulin and ochratoxin A, two abundant mycotoxins, by lactic acid bacteria[END_REF], fumonisins (Niderkorn et al., 2009), and patulin [START_REF] Fuchs | Detoxification of patulin and ochratoxin A, two abundant mycotoxins, by lactic acid bacteria[END_REF][START_REF] Wang | A new insight into the adsorption mechanism of patulin by the heat-inactive lactic acid bacteria cells[END_REF].

Numerous LAB (Lactobacillus rhamnosus, L. plantarum, L. casei, L. curvatus, L. coryniformis, L. brevis, L. mucosae, and L. pentosus) exhibit mycotoxin reduction ability in which binding is their main mechanism. [START_REF] El-Nezami | Binding Rather Than Metabolism May Explain the interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative α-Zearalenol[END_REF][START_REF] El-Nezami | Chemical moieties and interactions involved in the binding of zearalenone to the surface of Lactobacillus rhamnosus strains GG[END_REF][START_REF] Long | Removal of zearalenone by strains of Lactobacillus sp. isolated from rumen in vitro[END_REF]. The cell wall of LAB has the typical Gram-positive structure made of a complex assemblage of glycopolymers and proteins. It consists in a thick peptidoglycan that surrounds the cytoplasmic membrane and is decorated with proteins, polysaccharides, and teichoic acids [START_REF] Chapot-Chartier | Cell wall structure and function in lactic acid bacteria[END_REF][START_REF] Delcour | The biosynthesis and functionality of the cell-wall of lactic acid bacteria[END_REF]. The sugars and amino acids in glycopolymer or protein structures onto bacterial cell wall is different in each species of bacterial cell [START_REF] Chapot-Chartier | Cell wall structure and function in lactic acid bacteria[END_REF]. Moreover, polysaccharides have been suggested to be the important elements responsible for the binding of aflatoxin B1 [START_REF] Haskard | Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG[END_REF] and fumonisins (Niderkorn et al., 2009) by LAB. Polysaccharides in Gram-positive bacteria can be found in 3 parts: peptidoglycan, teichoic acids, and natural polysaccharides which include exopolysaccharides, capsular polysaccharides, and cell wall polysaccharides.

Bacterial polysaccharides exhibit great diversity, not only in sugar composition but also in linkage, branching, and substitution [START_REF] Chapot-Chartier | Cell wall structure and function in lactic acid bacteria[END_REF]. This means that peptidoglycan and teichoic acids are also related to mycotoxin reduction and the difference in mycotoxin removal capacity in each bacterial strain arises from the diversity of polysaccharides. [START_REF] Hernandez-Mendoza | Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria[END_REF] exhibited that the protoplast of Lactobacillus reuteri and L. casei Shirota also showed the capacity to reduce aflatoxin B1 in vitro. Both data implied that lipids were one of the components which were able to attach to the mycotoxin. All of these studies supported that the mycotoxin removal by bacterial cell was strain specific because of the diversity of each element structures onto bacterial cell wall, which includes proteins, polysaccharides, and lipids. Therefore, this class of microorganism has demonstrated great potential in fungal inhibition and mycotoxin removal in fermented legumes food products.

Fermentation against chemicals from agriculture

The presence of chemicals and pesticides in plant products is a growing societal concern. Some studies point out that following recommendation to increase the dietary uptake of fruit and vegetables means often increasing the uptake of chemicals. For instance in Brasil, the cumulative intake of organophosphorus and carbamate pesticides by high consumers of fruits and vegetables has been evaluated to represent up to 169% of the Acute reference dose (ARfD) [START_REF] Caldas | Exposure to toxic chemicals in the diet: Is the Brazilian population at risk?[END_REF]. This type of data gives rise to a debate whether benefits of eating vegetarian products are higher than risks (Richter ED and N. 2002). In any case, there is a consensus that the level of contamination by pesticide has to be significantly decreased.

However, despite several Integrated Pest Management (IPM) programs, decreasing the use of pesticides in agriculture and therefore in food products is difficult. One transient possibility (before an agricultural control of persistent organic pollutants) is to degrade pesticides or chemical degradation products through fermentation.

Considering the diversity of the earth microbiome, many chemicals can be transformed.

However, when limiting the diversity to the safe bacteria that can be encountered in fermented products, the choice of biocatalytic activities is narrowed. Degradation of pesticides during fermentation has already been analyzed in several studies showing a good potential of degradation of organophosphorus insecticides like chlorpyrifos during the fermentation of Kimchi by strains belonging to L. mesenteroides, L. brevis, L. plantarum, and L. sakei [START_REF] Cho | Biodegradation of Chlorpyrifos by Lactic Acid Bacteria during Kimchi Fermentation[END_REF]. Lactobacillus brevis was also seen as an active catalyst against the same family of products during the fermentation of milk products (Zhang et al., 2014). The degradation of organochlorine pesticides has also been investigated in milk during yogurt and cheese production showing the effect of starters [START_REF] Duan | Residue behavior of organochlorine pesticides during the production process of yogurt and cheese[END_REF]. Other examples show the capability of Micrococcus varians to degrade DDT to DDD and lindane to 2,4-, 2,5-, 2,6-and 3,4-dichlorophenol; 2,3,4-and 2,3,5-trichlorophenol; hexachlorobenzene; and pentachlorophenol. However in sausage, the degradation was only of 10 to 18% during the 72 h of sausage fermentation (Abou-Arab, 2002).

With the use of soy from the world market (mainly glyphosate resistant genetically-modified soy) to manufacture fermented soy in South-East Asia, it is likely that glyphosate is a main herbicide that has to be degraded although it can be combined with several other chemicals.

The screening of strains able to degrade pesticides from fermented food is thus a potential way to decrease the contamination of foods by chemicals. Up to now, this subject of bioremediation of pesticides has been more focused on environmental microbiomes [START_REF] Zhan | Recent advances in glyphosate biodegradation[END_REF] and the screening can also be extended to environmental lactic acid bacteria strains as in the following example for which Lactococcus lactis subsp. lactis able to degrade dinitrotoluene isomers has been isolated from earthworm intestine [START_REF] Shin | Anaerobic biotransformation of dinitrotoluene isomers by Lactococcus lactis subsp. lactis strain 27 isolated from earthworm intestine[END_REF].

Conclusion

Lactic acid bacteria are more and more used as a biopreservation agent against the development of pathogens and spoilage microorganisms in food products. They have also a potential to metabolize many compounds that are present in legumes and should be avoided in food products (Figure 1). They can thus decrease antinutritional factors and allergens as well as chemicals and pesticides used for culture. They can eventually remediate the presence of toxins from other microorganisms like mycotoxins and can oxidize biogenic amines.

Although they already fulfill these roles in many fermented products, their role for bioremediation is still underutilized due to a lack of knowledge about precise genes and mechanisms involved. The screening of LAB functions helping to decrease the concentration of all these potentially toxic molecules is thus of great potential to improve food safety of products, not only fermented. Some questions remain, as for instance how to orient metabolism towards the targeted compounds? Can we find, in LAB diversity, strains able to degrade all types of compounds? Is the metabolization of toxic compounds sufficient to decrease significantly the risk?

Although decreasing contamination is the first food safety strategy, lactic acid bacteria show a great potential through bioremediation functionalities that can help to decrease consumers' exposition to many toxic compounds. Some strains have already been identified in fermented foods but the development of starters with these functionalities has still to be developed.

  classes. Class I (Lantibiotics), class II (Non-Lantibiotics) and class III (Big peptides) depending on their chemical and genetic characteristics. The antibacterial activity of nisin, the most studied lantibiotics, has been demonstrated against Listeria sp., Micrococcus sp. and sporulating bacteria such as Bacillus sp. and Clostridium sp.[START_REF] Parada | Bacteriocins from lactic acid bacteria: purification, properties and use as biopreservatives[END_REF]).Nguyen et al.(Nguyen, Elegado et al. 2009) isolated the LAB from nem chua and determined their antimicrobial activity against pathogenic and sporulating strains such as Bacillus cereus, L. plantarum JCM 1149, Listeria monocytogenes, E. coli, and Salmonella Typhimurium. Five strains NH3.6, NT1.3, NT1.6, NT2.9, and NT3.20 showed a broaden antimicrobial activity against both pathogenic Gram-positive B. cereus and L. monocytogenes, and gram-negative E. coli with the zone of inhibition ranging from 1,5-6 mm (D-d), however no inhibitory effect against S. Typhimurium was detected(Nguyen, Elegado et al. 2009). L. plantarum HA2, HA3, HA5, HA8 and HA9 and L. fermentum HA6, HA7 and HA10 isolated from Vietnamese fermented vegetables showed a strong and broad antifungal activity against 7 out of 11 indicator mold and yeast strains (Aspergillus terreus, A. fumigatus, A. niger, Absidia corymbifera, Paecilomyces lilacinus, Geotrichum candidum, Fusarium sp., Scopulariopsis brevicaulis, Curvularia lunata, Penicillium sp., and Candida albicans)[START_REF] Ho | Lactobacilli and dairy propionibacterium with potential as biopreservatives against food fungi and yeast contamination[END_REF]).

  fermented dry-cured meat product (salchichón) with oregano essential oil used as surface mold inhibitor. MeatScience, 89, 35-44. Mills, E. N. C., Sancho, A. I., Rigby, N. M., Jenkins, J. A., Mackie, A. R. (2009). Impact of food processing on the structure and allergenic properties of food allergens. MolecularNutrition & Food Research, 53, 963-969. Motarjemi, Y., & Nout, M. J. (1996). Food fermentation: a safety and nutritional assessment. Joint FAO/WHO Workshop on Assessment of Fermentation as a Household Technology for Improving Food Safety. Bulletin of the World Health Organization, 74(6), 553. Nazzaro, F., Fratianni, F., Coppola, R., & Feo, V. D. (2017). Essential oils and antifungal activity. Pharmaceuticals (Basel, Switzerland), 10, 86. Nguyen, H., Elegado, F. B., Librojo-Basilio, N. T., Mabesa, R. C., & Dizon, E. I. (2009). Isolation and characterisation of selected lactic acid bacteria for improved processing of Nem chua, a traditional fermented meat from Vietnam. Beneficial Microbes, 1, 67-74. Niderkorn, V., Morgavi, D. P., Aboab, B., Lemaire, M., & Boudra, V. (2009). Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria. Journal of Applied Microbiology, 106, 977-985.Nowak-Wegrzyn, A., & Fiocchi, A. (2009). Rare, medium, or well done? The effect of heating and food matrix on food protein allergenicity. Current Opinion inAllergy and Clinical Immunology, 9,[234][235][236][237]

Acknowledgements: This work was funded by the Erasmus+ AsiFood «Universities as key partners for the new challenges regarding food safety and quality in ASEAN» project, the Bualuang ASEAN chair professorship of Thammasat University and the French Embassy in Thailand. The author are thankful to Dr Giorgia Perpetuini for her active participation to the redaction of this text from bibliography on biogenic amines to the editing of the final manuscript, and to Dr Guillaume Da for his precious help to establish the network.