G Balaj

Z Tamanai-Shacoori

D Olivier-Jimenez

A Sauvager

M Faustin

L Bousarghin

S David-Le Gall

S Guyot

D Nebija

S Tomasi

M L Abasq

and green tea Flavones

Keywords: Catechin Epicatechin Apple, apricot, peach, grape, berries, cereals, chocolate, red wine, nuts, black Parsley, celery, lettuce, artichoke, herbs (rosemary, thyme, oregano, etc.), citrus fruits, cereal grains, sweet peppers Flavonols Quercetin Kaempferol Myricetin Isorhamnetin Yellow and red onion, caper, lettuce, parsley, berries, green Soybean, tofu, green bean, lentil, chickpea, pea, mung bean, broad bean, medicinal herbs Anthocyanidins

Microbiota is known to play a pivotal role to generate bioavailable and bioactive low molecular weight metabolites from dietary polyphenols. 5-O-caffeoylquinic acid (5-CQA), one of the main polyphenols found in diet, was submitted to a resting cells biotransformation study using three gut bacteria species Lactobacillus reuteri, Bacteroides fragilis and Bifidobacterium longum. These bacteria were selected in accordance to their belonging to the main phyla found in human gut microbiota. Our study highlighted the ability of only one of the strains studied, L. reuteri, to bioconverse 5-CQA into various metabolites due to the expression of cinnamoyl esterase enzyme as the first step. Interestingly, one natural known compound, esculetin, was for the first time described as a 5-CQA derived metabolite after conversion by a gut bacterium, the other metabolites were already reported. This evidence put in the spotlight an interesting oxidative pathway occurring in vivo by intestinal microbiota affording to esculetin. This molecule was also identified after electrochemical and enzymatic oxidations of caffeic acid.

VI

List of figures

Figure 5. Representative scheme of absorption and metabolism pathway of 5-CQA in humans (I) almost one-third of 5-CQA intake is absorbed in upper digestive (stomach and/or small intestine, without hydrolysis and then enter to the circulatory system (II), a small amount of the 5-CQA (around 7%) is hydrolysed into CA and QA and then absorbed via the small intestine. (III) the major part reaches the colon and is metabolised by gut bacteria; (IV) 5-CQA as native form and its metabolites are absorbed and/or are subjected to liver metabolism, including methylation, by COMT = catechol-Omethyltransferase (COMT), glucuronidation (GT) or sulfation (ST), [START_REF] Lu | Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action, and future directions[END_REF] ...................... 21 Figure 6. An overview of the biological activities of 5-CQA in humans [START_REF] Lu | Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action, and future directions[END_REF]................... 22 Figure 7. A schematic covering the microbiome nomenclature, highlighting the microbiota and their "theatre of activity" [START_REF] Berg | Microbiome definition re-visited: old concepts and new challenges[END_REF] Figure 11. The Network between gut bacteria and innate and adaptive immune system [START_REF] Sansonetti | The innate signaling of dangers and the dangers of innate signaling[END_REF]

X

List of tables

General Introduction

Polyphenols, natural secondary metabolites of plants, have drawn increasing attention from the scientific community due to their ability to decrease oxidative stress involved in many pathologies and their positive effects in modulating redox cellular signalling pathways (Zhang and Tsao, 2016). However, given that they are characterized by low bioavailability and considering the crucial role of microbiota in generating bioavailable and bioactive metabolites (Pasinetti et al., 2018), further insights are focused nowadays on their gut-derived metabolites as a promising approach to prevent and attenuate many pathologies such as cancer, diabetes, In this context, our research focused on one of the main dietary phenolic compounds, 5-O-caffeoylquinic acid (5-CQA), also called chlorogenic acid. The objective was twofold: 1)

to study the reciprocal interactions between this compound, 5-CQA, and three intestinal bacteria Lactobacillus reuteri, Bacteroides fragilis and Bifidobacterium longum, which are representative of the major phyla found in the gut; 2) to provide information on the potential effects of 5-CQA and its gut-derived metabolites on gut health.

In Chapter I, state-of-the-art scientific literature on polyphenols and the gut microbiota is presented. Since polyphenols have been widely reviewed, a brief bibliographic presentation is provided. Next, we present an overview of the gut microbiota which represents one of the main topics in the last decade. Finally, we discuss how the synergistic interactions between gut microbiota and polyphenols can alleviate brain disorders.

In Chapter II, the results of our study are presented, divided into subsections. The first part consist of electrochemical and enzymatic studies performed on 5-CQA and one of its gut-derived metabolites, caffeic acid (CA). Then, the biotransformation process of 5-CQA carried out by mono-and co-culture using the resting cell method is presented. Furthermore, we will describe how we have investigated the potential effect of 5-CQA, when supplemented, to modulate the growth and metabolic profiles of L. reuteri and B. fragilis. Finally, using an original in vitro quadruple cell model, we describe the potential impact of 5-CQA and its gutderived metabolites on modulating gut function as a target strategy to provide positive health effects.

The discussion and opening of new perspective are elaborated in Chapter III. Finally, the materials and methods section are detailed in the last chapter (IV) of this manuscript. Polyphenols consists of at least one aromatic ring, containing one or more hydroxyl substituents (Figure 1). Indeed, they can range from simple phenolic acids (containing single rings) to highly polymerized compounds such as hydrolysable and condensed tannins (see The most phenolic compounds occur naturally as conjugates with sugars or as esters with organic acids [START_REF] Manach | Polyphenols: food sources and bioavailability[END_REF]. More rarely, they are found as free molecules (aglycones). Owing to their structural diversity, a wide range of phenolic compounds is found in nature. Currently, more than 8000 phenolic compounds have been identified The non-flavonoid polyphenol group consist of phenolic acids, stilbenes, lignans and hydrolysable tannins. The main dietary ones and their structures are shown in Figure 2.

Chemically, phenolic acids have at least one aromatic ring in which at least one hydrogen atom is substituted by a hydroxyl group. They are divided into two major groups: hydroxybenzoic acids (HBAs) (gallic, protocatechuic, syringic and vanillic acids) and hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acids). Hydroxybenzoic acids are mainly present in plant food as conjugates, but they can be also free in some fruits (e.g., gallic acid in persimmon)

or released during fruit or vegetable processing (Tomás-Barberán and Clifford, 2000). They constitute a smaller portion of edible plants and generally form components of a complex structure such as hydrolyzable tannins. An example of conjugated HBAs is gallic acid (tannin), which can be a dimer, trimer or tetramer (ellagic, tergallic and gallagic acids, respectively).

Tannins consist of hydrolysable tannins (gallotannins and ellagitannins) and non-hydrolysable, condensed tannins (proanthocyanidins), which are derivatives of flavonoids (Figure 2).

Strawberries, pomegranate and walnuts are good dietary sources of ellagic acid. Gallotannins, unlike ellagitannins, are rarely found in plant foods, and occur almost exclusively in mango, chestnuts and red sword beans [START_REF] Manach | Polyphenols: food sources and bioavailability[END_REF].

Hydroxycinnamic acids can be found in plants in their free and esterified forms e.g., with quinic acid (chlorogenic acids) or as glycosides (e.g with glucose in 1-O cinnamoylglucose and with choline e.g., in sinapine). Coffee and some vegetables, fruits, and cereals are rich sources of hydroxycinnamic acids [START_REF] Crozier | Dietary phenolics: chemistry, bioavailability and effects on health[END_REF].

Lignans are commonly found as glycosides in many foods. Secoisolariciresinol, the main representative and its gut-derived metabolites enterolactone and enterodiol, have attracted the attention of scientists due to their potential biological effects ( Flavonoids are conjugated with mono-and polysaccharides (glucose, galactose, rhamnose, apiose), linked by β-glycosidic bonds to a hydroxyl group (O-glycosides) or to a carbon atom of the aromatic ring (C-glycosides). The main food sources are listed in Table 1.

Table 1. The main flavonoids and their food sources (Tomás-Barberán et al., 2020)

Flavonoid class Main compounds Food sources

Flavanols

According to a study conducted by the European Prospective Investigation into Cancer and Nutrition (EPIC), the average intake of polyphenols in a healthy diet is 1 g/day. The most abundant polyphenols were phenolic acids (52.5-56.9 %), with chlorogenic acids being the most prominent compounds in this class, except in men from Mediterranean (MED) countries and in the United-Kingdom (UK) health-conscious group where the main compounds are flavonoids (49.1-61.7 %) (mainly flavanols (16-29%)). On the other hand, stilbenes and lignans are present to a lesser extent with percentages of 0.1-0.5% and 0.1-0.7%, respectively.

5-O-caffeoylquinic

I.A.2. Biological activities of polyphenols

It is well known that oxidative stress is closely related to mitochondrial dysfunctions and is one of the main factors associated to many pathological conditions including neurological disorders, cancer, metabolic diseases and others [START_REF] Valko | Free radicals and antioxidants in normal physiological functions and human disease[END_REF]. We will first present the health risks of oxidative stress and ways to combat it, such as the use of polyphenols due to their properties. At low levels, ROS can function as signalling molecules that regulate basic cellular activities, such as cell growth and cellular adaptive responses (Sies and Jones, 2020). Under physiological conditions, there is a natural balance between the production of reactive species and the antioxidant defence system of the body. When this balance is compromised, it causes an accumulation of ROS, and when this can occur on a short time scale, it is known as acute stress and ends with a return to redox balance. However, when oxidative stress persists over time, the body fails to rebalance its redox status [START_REF] Lushchak | Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions[END_REF]. This causes so-called chronic, or pathological oxidative stress which can consequently lead to damage of cellular molecules such as DNA, proteins and lipids. There are two mechanisms through which

I.A.2.2. Antioxidant properties

The antioxidant effects of polyphenols are attributed to their chemical structure.

Polyphenols act as natural antioxidants thanks to their free radical-scavenging properties. They exert their antioxidant properties against reactive oxygen species (ROS) via hydrogen atom transfer (HAT) or electron transfer (ET) mechanisms. In the first mechanism, the antioxidant ArOH can give an hydrogen atom to a free reactive radical (R • ) leading to ArO

HAT (H-atom tranfer)

R • + ArOH → R -H+ ArO •
In the second mechanism, the antioxidant can donate an electron to the free radical, which becomes a cation radical. In this mechanism, the lower the ionization potential (IP), the easier the electron abstraction, which means a higher antioxidant activity. This electron transfer mechanism will be deeply developed in electrochemistry part.

Electron transfer

R • + ArOH → R-H -+ ArO •+
The antioxidant potential of polyphenols is related to their structure. For example, in the case of flavonoids, the number and positions of the hydroxyl group (-OH), the presence of a double bond (C2=C3), glycosylation and the presence of substituents in the rings are the main factors that determine their antioxidant potential. It was revealed that the presence of the 3-OH group contributed to the increase of antioxidant properties compared to those with less OH groups in the ring. Thus, quercetin is considered as a strong antioxidant compound. The superior antioxidant properties are also related to the presence of a C2=C3 double bond in conjugation with the C4-carbonyl group. When considering glycosylation, the C-glycoside form has demonstrated greater antioxidant ability than the O-glycoside in chemical assays (T.

Wang et al., 2017).

In the case of phenolic acids, a longer distance separating the carbonyl group and the aromatic ring of the phenolic acid, appears to increase antioxidant activity. For example, in the case of cinnamic acid and its derivatives compared to benzoic acids, hydroxyl groups on the benzoic ring at the ortho-position and/or para-position can lead to high antioxidant activity compared to those with other positions and to unsubstituted phenol (Zhang and Tsao, 2016).

I.B.2.3. Modulators of signalling pathway

Another mechanism of oxidative stress suppression by polyphenols is modulation of multiple signalling pathways, such as mitogen-activated protein (MAP) kinase and NF-κB, thus, suppressing the release of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, and IL-1)

(

I.A.3. Chlorogenic acids compounds

We have focused our study on 5-O-caffeoylquinic acid, known as chlorogenic acid, one of the main phenolic compounds in the daily diet. Indeed, chlorogenic acids are a group of compounds formed by the esterification of trans-cinnamic acids such as caffeic acid (CA), pcoumaric acid or ferulic acid and quinic acid (Clifford, 2000). Depending on the type of ester substituent, the most common chlorogenic acids are caffeoylquinic acid (CQA), dicaffeoylquinic acid (diCQA), p-coumaroylquinic acid (p-CoQA), feruloylquinic acid (FQA)

and caffeoylferuloylquinic acid (CFQA) (Figure 3) (Lu et al., 2020). The former 3-CQA is referred to neochlorogenic acid (Figure 4). 5-CQA also exhibits geometric isomerization in the cis and trans forms (Clifford et al., 2017). Many plants, including medicinal plants, fruits and vegetables produce this compound.

For example, in coffee beans (100 mg/g) or green coffee beans it represents 76 to 84% of total CGAs. Its content varies in fresh ground coffee (8.19 to 23.78 mg/200 mL) or instant coffee (9.45 to 41.05 mg/200 mL) [START_REF] Lu | Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action, and future directions[END_REF]. Moreover, its content also depends on the coffee variety (Arabica vs. Robusta) and roasting time (Table 2). Indeed, coffee is the main dietary source of 5-CQA (Farah and de Paula Lima, 2019). In addition, it has been reported that cider contains a high amount of 5-CQA [START_REF] Guyot | Multiplicity of Phenolic Oxidation Products in Apple Juices and Ciders, from Synthetic Medium to Commercial Products[END_REF]. Additionally to coffee and cider, fruits are also good dietary sources of 5-CQA including apples (0.41 to 1.16 mg/g), pears, plums, cherries [START_REF] Crozier | Dietary phenolics: chemistry, bioavailability and effects on health[END_REF]. Among vegetables, eggplant (1.4 to 28.0 mg/g), carrot (0.3 to 18.8 mg/g), artichoke (1.1 to 1.8 mg/g), and pepper (0.7 to 0.9 mg/g) also contribute to the dietary intake of 5-CQA (Lu et al., 2020).

Table 2. 5-CQA content in Arabica vs Robusta, compared to total chlorogenic acids and effects of roasting time on this content (Lu et but also to better understand the mechanism of action of 5-CQA in vivo, which will be of interest for the development of new nutraceutical uses of this compound.

Although the mechanism by which 5-CQA exhibits its beneficial effects has not been fully elucidated, it has been suggested that it mainly involves its free radical scavenging properties but also its targets in multiple signalling pathways, thus regulating transcription factors and protein kinases. Interestingly, [START_REF] Amano | Safety Pharmacological Evaluation of the Coffee Component, Caffeoylquinic Acid, and Its Metabolites, Using Ex Vivo and In Vitro Profiling Assays[END_REF] 2019). The differentiation between the microbiome and microbiota terms are depicted in Figure 7.

Figure 7. A schematic covering the microbiome nomenclature, highlighting the microbiota and their "theatre of activity" [START_REF] Berg | Microbiome definition re-visited: old concepts and new challenges[END_REF] As a key component of microbiome, over the last years, bacteria are in the spotlight of microbiome research. The number of bacteria was initially estimated to outnumber human cells

I.B.2.3. Drug-gut microbiota interactions

Some studies have shown that the oral antibiotics additionally to their action on targeted microbes, also affect the composition and functions of the entire intestinal microbiota, consequently they can impact host health [START_REF] Froehlich | Cognitive impairment by antibiotic-induced gut dysbiosis: Analysis of gut microbiota-brain communication[END_REF][START_REF] Hagan | Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans[END_REF]Maier et al., 2021;[START_REF] Maurice | Xenobiotics shape the physiology and gene expression of the active human gut microbiome[END_REF][START_REF] Mu | Antibiotic effects on gut microbiota, metabolism, and beyond[END_REF]. In addition the antibiotics, growing evidence that other drugs also affect the intestinal microbiota [START_REF] Maier | Extensive impact of non-antibiotic drugs on human gut bacteria[END_REF][START_REF] Vila | Impact of commonly used drugs on the composition and metabolic function of the gut microbiota[END_REF], highlighting the importance of investigating the effects of drugs on the composition and metabolic function of the gut microbiota (Guthrie and Kelly, 2019).

From a pharmacological point of view, the stomach is not favourable absorbing environment for most orally administered drugs. Therefore, they pass into the small intestine and following their absorption undergo pharmacokinetic processes of distribution, metabolism, and excretion. In addition, some drugs, including drugs poorly absorbed in the small intestine and modified release formulations, can reach the colon The human brain constantly communicates with the gut in a bidirectional complex way, known as the gut-brain axis. However, due to the key regulatory role of microbiota on this axis, the microbiota-gut-brain axis is emerging as a concept in the field of neuroscience. These potential connections between the gut microbiota and the brain, include neuronal, immune, endocrine, and metabolic signalling (Cryan et al., 2019b), which will be developed in subsequent headings.

I.B.3.1. Microbiota-brain immune signalling

One of the main routes that link the gut and brain is through immune mechanisms (Fung, 2020). The largest and the most complex immune system is found in the gut and is known as the gut-associated lymphoid tissue (GALT). GALT represents 70-80% of the body's immune cells, which constantly communicate with the gut microbiota, in turn influencing and shaping both the immune system at the intestinal and systemic levels (Kasper et al., 2019).

The key role of GALT is to provide immune tolerance to commensal microorganisms while initiating immune responses against pathogenic ones (Mörbe et al., 2021). The intestinal epithelium separates and limits the contact of bacteria with the lamina propria, thus providing the defense against invading microbes and external factors. (Chelakkot et al., 2018;Hooper, 2015). Most of the GALT`s organization in the colon is the same as that in the small intestine.

Moreover, the colon has a dual mucus layer, with a thick largely sterile inner layer and a thinner outer layer. Below the gut epithelium, a layer of connective tissue called the lamina propria is located, where various immune cell types (e.g., dendritic cells, macrophages, and T and B lymphocytes) can be found (Cader and Kaser, 2013). The GALT organization of the colon is shown in Figure 10.

I.C.3.4. Neuronal pathway

The gut has 200-600 million neurons known as enteric nervous system (ENS). This system can function autonomously although it communicates with CNS via parasympathetic and sympathetic branches of the autonomous nervous system (ANS). The scientists analyzed the fecal microbiota of 1054 Belgian people and validated the results in an independent cohort of 1,063 individuals from the Dutch LifeLines DEEP (LLD) and casecontrol studies previously published. These researchers found that the genera Dialister and

Coprococcus were depleted in people with depression despite antidepressant treatments.

Furthermore, the scientists described the correlation between a higher quality of life and the abundance of Faecalibacterium and Coprococcus. This study also revealed the ability of gut microbiota to produce particular neuroactive metabolites associated with mental health Beneficial effects of polyphenols can be also related to their ability to shape gut microbiota in favour to other specific gut microbial species that can provide health benefits.

For instance, Lactobacillus and Bifidobacterium are known for their contribution in gut barrier function, supressing proinflammatory cytokines and even inhibiting pathogenic bacteria HAT (H-atom tranfer)

ArO -H + R • → ArO • + R -H Eq 1
SET -PT (Sequential electron transfer-proton transfer)

ArO -H + R • → ArOH •+ + R -→ ArO • + R -H Eq 2
SPLET (sequential proton loss-electron transfer)

ArO -H → ArO -+ H + ArO -+ R • → ArO • + R -and R -+ H + → R-H Eq 3
The SET mechanism leads to a reduction of the oxidized substrate in two steps: a first electron transfer step forms the cation radical ArOH •+ (Eq 2). However, this reaction mechanism is unlikely between phenolic compounds and radical oxygen species (ROS) due to the high instability of the ArOH A third process involves the simultaneous transfer of an electron and a proton SPLET (Sequential Proton-Loss Electron Transfer) (Eq 3). This mechanism was notably observed during the study of the trapping of the DPPH • radical. The balance will be the same as during the HAT hydrogen atom transfer, but the sequential process involving the phenolate anion favors higher rate constants, because the ArO -phenolate is a better electron donor than phenol

II.A.1.2. Potential Redox

In the case of an oxidation reduction reaction:

Ox + ne -⇌ Red E °
where Ox and Red represent the oxidized and reduced forms, and n the number of exchanged electrons.

When a substance loses an electron, its oxidation state increases; thus, it is oxidized.

When a substance gains an electron, its oxidation state decreases, thus being reduced.

The In our biotransformation study, it would be interesting to explore metabolites that might result from similar oxidation mechanisms.

Hence, in this study, the objective was twofold. Electrochemical and enzymatic oxidations of 5-CQA and CA were performed with the aim to identify the oxidized compounds.

The knowledge of the MS/MS data of the 5-CQA oxidized molecules obtained from the oxidation processes will facilitate the search of these molecules in biotransformed extracts by comparison of their chromatographic and MS/MS patterns.

II.A.2. Results and discussion

Solution of 5-CQA (2 mM) and CA (2 mM) were prepared in PBS (pH 5.8) with the aim of studying whether specific molecules of interest could arise from their oxidation via an electrochemical method. In a first step, the cyclic voltammetry was performed to access the anodic potential of 5-CQA and CA. In a second step, the electrolysis of the two phenolic compounds were performed by a chronoamperometry technique (i = f (t)) at a fixed potential during 20 min. The electrolyzed samples were then analyzed by LC-MS 2 . For a better understanding of the mechanism involved, an enzymatic process was also carried out.

Comparison of the resulting chromatographic profiles will allow the identification of the oxidized products. for 5-CQA and CA). However, it can be noted here that under these experimental conditions at pH 5.8, the system is not electrochemically reversible, which is indicated by the large separation of the potential peaks (E = Epa -Epc) and by the current ratio ic/ia, which is less than 1, attesting to the instability of ortho-quinone. The peak (7) at RT= 19.35 min corresponds to the residual 5-CQA, for both methods used. Indeed, it was easily identified based on spectral patterns (RT, UV λmax at 325 nm), and mass spectrum (m/z 353) and confirmed by comparison with an analytical standard. The comparison of LC-MS 2 chromatograms of 5-CQA, before and after electrolysis, highlighted the detection of numerous peaks, corresponding to oxidized metabolites of 5-CQA (Figure 25). The majority of them corresponded to ions with m/z 705 (peaks 2, 5, 6, 10-24). In addition, one peak 1 at m/z 703 eluted before 5-CQA, at RT =8.58 min. Finally, peaks 3, 4 and 25 corresponded with ions at m/z 723. We can note that most of the products formed by oxidation were less polar compounds than 5-CQA, except peaks 1-6. The same ions were detected after oxidation (Figure 25 and Table 3). However, we noted that the enzymatic oxidation of 5-CQA showed slight differences compared to the electrochemical oxidation. Thus, even if peaks 14 and 15 were the most abundant compounds with the two methods, their relative intensity were higher than the control 5-CQA after 5-CQA According to Guyot et al, the mechanism of enzymatic oxidation of 5-CQA, led to the formation of dimers. As a first step, CQA is oxidized in ortho-quinone, which is not stable and reactive. Then these ortho-quinones are involved in further oxidation reduction reactions and chemical addition. All these reactions lead to the formation of secondary o-quinones which can react with primary o-quinones and give semi-quinones (radical form). Quinones and semi quinones can react and give a large number of products by addition, dimerization or water addition followed by intramolecular rearrangement. Polymerization seems to be minor (Guyot and Poupard, 2011).

By comparison of the LC-MS 2 data, the same oxidized molecules than those identified by Guyot's team, enzymatically, were detected by the electrochemical study of 5-CQA and were confirmed by spectral patterns comparison (Table 3). The UV spectra of these oxidation products were in accordance with those previously obtained by enzymatic oxidation of 5-CQA.

In addition, MS/MS spectra were similar to previously data published by Guyot et corresponding to hydroxylated species, could arise from nucleophilic addition of water to CA derivatives coming from typical coupling reactions. In a same manner, the peak 7 with m/z 375 could be originated from a water addition to a CA dimer. Peaks 10 and 11 correspond to trans and cis CA geometric isomers, respectively, with both a specific MS/MS fragmentation corresponding to a decarboxylation (ion at m/z 135). From cis-CA, with a mechanism which will be described in Biotransformation section, comes the ion at m/z 177, which was identified as esculetin (peak 8). Peaks 8, 10 and 11 were also identified after enzymatic oxidation.

Nevertheless, CA dimers with m/z 357, previously identified and formed by either a Michael- Further purification steps will be interesting to carry out in order to determine their structures. An ion with m/z 137 was detected at RT 15.33 nm and corresponds to a more polar compound than CA. In accordance to its max at 280 nm, this compound has lost the side chain double-bond conjugation and could come from decarboxylation of CA then reduction, leading to its identification as 4-ethylcatechol. This compound has been previously reported to be produced by L. johnsonii (Bel-Rhlid et al., 2013). Finally, peaks 3 and 4 characteristic of the enzymatic study have shown m/z 251 and 265, respectively with a RT close to each other. In the same manner than previously, further purification steps will be required in order to determine the structures of these compounds. Often, the approach used to identify Bifidobacteria involves the presence of a fructose-6-phosphoketolase (F6PPK) activity, a key enzyme of the genus Bifidobacterium.

Bifidobacteria coexist with a large variety of bacteria in the human intestinal microflora The oxidation capacity of L. reuteri led to less diverse metabolites in comparison to those obtained either electrochemically and enzymatically where dimers and trimers were reported.

Thus, esculetin may exert interesting and beneficial biological effects on gut microbiota, which must be further evaluated. Novel synbiotics could be formulated from the association of L.

reuteri with chlorogenic acids.

II.B.2. 1. Introduction

Polyphenols are known for their broad range of positive bioactivities in humans. These Specific biotransformed metabolites appeared in all the clusters but mostly as self-looped nodes. We then annotated MN by dereplication using GNPS library. Green nodes depicting metabolites derived from 5-CQA bioconversion by L. reuteri and showing a cos > 0.9 were displayed in Figure 34. Among them cis-CA, trans-CA, coumaroyl quinic acid and esculetin caught our attention linked to their structure close to 5-CQA. At this stage, we must underline that MN analysis led to the annotation of two nodes (labelled by an asterisk in Figure 33) and To validate some of these annotations, various analytical standards were injected as depicted in Table 5 and led to the identification of 5-CQA (ID 3), esculetin (ID 4) and trans-CA (ID 5). Surprisingly, both cis and trans geometric isomers of CA were identified in MN.

To our knowledge, it is the first report of the presence of these two CA isomers after 5-CQA biotransformation either by bacteria or human gut microbiota. Recently Clifford expressed that only few studies were reported on metabolism of cis isomers in humans (Clifford et al., 2020).

It is to note that MN analysis identified cis-CA at RT: 21.5 min highlighting a misidentification of this standard in GNPS library. Our results concerning the attribution of ID 5 and 8 as respectively trans-CA and cis-CA are in line with those previously published (Oniszczuk,

2016

). The main point to highlight in our MN (Fig. 2) is the annotation of [M -H] -ions at m/z 177 as being esculetin. While Mortelé et al. (Mortelé et al., 2019) attributed this ion as caffeoyl quinone, we confirm the identification of esculetin using analytical standard (Table 5). for which we have found a sequence similarity with an ubiquinol oxidase in the genome of L.

Esculetin was reported only once time as an in vivo metabolite

It is interesting to cite that [START_REF] Parrino | Photochemical and photocatalytic isomerization of trans-caffeic acid and cyclization of cis-caffeic acid to esculetin[END_REF] reported the no formation of esculetin at pH 3.5 in comparison to the possible conversion with pH > 6.30. These data suggest that esculetin can not be formed in stomach. To confirm the ability of L. reuteri to produce esculetin from CA after 5-CQA biotransformation, we used BHI instead of PBS at pH 5.8 in order to prevent CA autooxidation. This phenomenon was detected after CA incubation at 37°C in PBS at 5.8 but in a lesser degree than in biotransformed extracts (see Figure 37). CA autooxidation has not been checked so far in previous studies of 5-CQA and CA biotransformation. In our study, we confirm that L. reuteri was able to produce esculetin either from 5-CQA and CA in higher amount when compared to any controls. Moreover, the absence of cis-5-O-caffeoylquinic acid (peak 3) after 5-CQA incubation in BHI (Figure 37) confirm the major formation of cis-CA by L. reuteri from isomerisation of trans-CA.

b) Characterization of oxidized compounds of 5-CQA and CA by electrochemical and enzymatic methods

For a better understanding of the oxidative biotransformed pathway leading to the esculetin formation, we have performed electrochemical and enzymatic oxidation of 5-CQA and CA.

b-1) Electrochemical studies of 5-CQA and CA

The electron transfer properties of the two dihydroxycinnamic acids, 5-CQA and CA, were investigated by cyclic voltammetry (CV) in 0.1 M PBS at pH 5.8, similar conditions to those of biotransformation studies. As expected, for both compounds, the ortho-diphenol function is oxidized in a two-electron-proton mechanism to form an ortho-quinone (visible on the anodic scan at Epa = +0.55 V and at Epa = +0.38 V, respectively for 5-CQA and CA), which is reduced back to the catechol form on the reverse cathodic scan (Epc = + 0.11 V and Epc = + 0.14 V, respectively for 5-CQA and CA) (see Figure 23 as previously reported).

However, here, the system is not electrochemically reversible indicated by the large potential peak separation (Epa -Epc) and by the current ratio ic/ia, which is below 1, attesting the instability of the orthoquinone. The electrochemical oxidation mechanisms of dihydroxycinnamic acids have been well described and Hapiot et al reported that the CV electrochemical parameters show that the caffeoyl quinone becomes less stable from pH 4, even at the timescale of CV [START_REF] Giacomelli | Electrochemistry of Caffeic Acid Aqueous Solutions with pH 2.0 to 8.5[END_REF][START_REF] Hapiot | Oxidation of caffeic acid and related hydroxycinnamic acids[END_REF]. Then, controlled potential electrolysis of 5-CQA and CA were achieved in pH 5.8 0.1 M PBS in a modified UV cell at a platinum grid electrode. As a first step, UV-Visible spectra were recorded during the linear sweep voltammetry LSV at different potential values. Up to + 0.75 V and + 0.52 V, 5-CQA and CA remained under their native form and the wavelength of maximum absorption λmax were measured at 320 nm. Above these potentials, an absorption band was visible around 400 nm for both compounds, attesting their oxidation to their quinone form. With this knowledge, the electrolysis of 5-CQA and CA were conducted at + 0.8 and + 0.6 V, respectively. The electrolysis was stopped after 20 minutes (corresponding to an electrolysis yield of 15%), a time for which the quinone is still present in solution (λmax = 404 and 420 nm for 5-CQA and CA quinone respectively). All the electrolyzed samples were then analysed by LC-DAD-ESI-MS 2 .

b-2) Comparison of LC-DAD-ESI-MS 2 of biotransformed, electrolyzed and enzymatic samples.

CQA and CA were submitted to an enzymatic oxidation by PPO (Cheynier and Moutounet, 1992a) and directly analysed by LC-DAD-ESI-MS 2 . These data were compared to those of 5-CQA biotransformed and CA electrolyzed samples (Table 6). Esculetin was each time identified independently of the method used underlining the stabilization of the oxidative CA product, caffeoyl quinone, into its coumarin form, but in far lesser amounts compared to the biotransformation process. Unsurprisingly, no traces of caffeoyl quinone were found in any samples studied. The value of λmax were in accordance to those in previous report [START_REF] Tattini | Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: A fluorescence microscopy analysis[END_REF].

Analysis of electrolyzed and enzymatic sample of 5-CQA did not lead to the detection of esculetin confirming that the first step of production of this coumarin is the cleavage of bond ester which was not permitted under electrochemical and PPO conditions. Moreover, the comparison of LC-DAD-ESI-MS 2 analysis of the oxidized samples of 5-CQA and CA, obtained electrochemically and enzymatically, has demonstrated that no other oxidative pathway than the one leading to esculetin occurs by biotransformation. Indeed, for example for 5-CQA, the oxidation by the electrode or by the PPO also involves dimerization pathways.

Interestingly, the LC-MS/MS profiles of the electrolyzed and enzymatic solutions of 5-CQA were very similar, leading to dimers involving mechanisms described by [START_REF] Guyot | Multiplicity of Phenolic Oxidation Products in Apple Juices and Ciders, from Synthetic Medium to Commercial Products[END_REF]. The oxidation of CA enzymatically also led to dimers and trimers formation The first set of questions aimed to identify the capacity of individual gut bacteria to biotransform 5-CQA which is specific to one species. Considering the complex interactions between bacteria in the gut, we studied the biotransformation of 5-CQA when two bacteria, L. ). Various modes of interaction there have been described among microorganisms in the gut, such as mutualism, which is a win-win interaction that benefits both participants; commensalism, where the commensal benefits without the partner being helped or harmed. On the other hand, negative interactions could be present such as amensalism, where one organism can inhibit or destroy the other; competition, where both the participants compete for the same resources; and parasitism, corresponding to a relationship in which one pair benefits from the other microorganism (Faust and Raes,

2012).

Remarkably, in terms of bacteria-bacteria interactions, commensal bacteria regulate pathogen colonization and activity, thus preventing pathogenic infections. This is mainly mediated by colonization resistance as well as the ability to inhibit pathogen adhesion to human cells. The colonization resistance consist directly by various ways such as nutrient and territory competition, toxic substances, and gut metabolites production (e.g., through commensalspathogens/pathobionts interactions), or indirectly by modulating immune host functions and The interaction between bacterial strains, in which the metabolic by-products or endones produced by one species can be nutrient resources and used by others, is termed crossfeeding. Cross-feeding is a specific form of mutualism and can be mediated via several forms including metabolites, substrates, mutual and augmented cross-feeding as depicted in Figure 38 (Smith and Wissel, 2019). Indeed, the identification of specific gut microbes involved in the biotransformation of polyphenols into bioactive compounds, is crucial for a better understanding of the favorable impact of polyphenols on health. However, since the gut microbiota is a dynamic ecosystem, microorganisms do not function independently, but interact with each other and with the host.

To move to these types of strategies and use more practical models to understand the bacterial community at the laboratory scale, the cultivation of bacteria in the community in the form of co-cultures is used. Indeed, the complex cultivation of bacteria in co-culture and even isolation of gut bacteria in general, have impacted the limitations and available information related to this. Additionally, there is growing interest in co-culture engineering in the field of natural product discovery (Scherlach and Hertweck, 2009).

Although co-cultures still have limitations, especially in the case of anaerobic bacteria, they are a very useful approach to identify in vitro metabolic interactions between gut bacteria.

Hypothesis

We hypothesized that cross-feeding could be involved in the biotransformation process.

We have planned to investigate an anaerobic bacterial co-culture as a model of study based on the use of the two gut bacteria L. reuteri and B. fragilis. Based on our previous results, the cinnamoyl esterase capacity was only expressed by L. reuteri, and we questioned whether interactions between these bacteria can influence the biotransformation process. Indeed, we performed some studies by co-culturing L. reuteri and B. longum. However, because the former colonies weresimilar, it was difficult to distinguish them and we were not able to perform this study in a good manner because the time schedule of the thesis.

II.B.3.1. Results and discussion

With the goal to give information on bacterial interactions in the gut community and their effects on the biotransformation of 5-CQA, we performed the resting cell biotransformation study using a co-culture of B. fragilis and L. reuteri. As a first stage, the two selected strains were co-cultured, for 30 hours ((corresponding to the stationary phase (data not shown)), with an equivalent number of bacteria, based on CFU determination at T0 as 7.60 x 10 7 CFU.mL -1 and 8.14 x 10 7 CFU.mL -1 for L. reuteri and B. fragilis, respectively. In parallel, the two bacteria were grown alone, which helped us to compare their culture evolution profile with those obtained in the co-culture. Then, we evaluated the impact of interactions between the two bacterial species on the biotransformation of 5-CQA. As described previously, biotransformation was performed using the resting cell method. When the stationary phase was reached (at T30), 5-CQA (1 mM) was added on the pellet obtained after co-culture or mono-culture (L. reuteri alone). With all negative and blank controls, they were then incubated under anaerobic conditions using sealed jars and anaerobic bags for 24 h. Finally, the supernatant and pellet were separated and treated with ethyl acetate to obtain two extracts (SN and pellet) which were further subjected to LC-UV-ESI-MS 2 . Herein we present the results obtained on SN extracts as the metabolites are less concentrated in pellet ones. First studies have been undertaken and should be further investigated. The concentrations of metabolites found in each extract are shown in Figure 42. We have observed that not equivalent amount of 5-CQA, CA and esculetin were formed when the two modes of biotransformation were compared, in mono or co-cultures.

Interestingly, esculetin was found at higher amounts (438.4 µg/mL) in SN extracts after coculture of L. reuteri and B. fragilis compared to esculetin formed when L. reuteri is alone (353.9 µg/mL) corresponding to a 23.9 % of increasing. Contrary, trans-CA was found to be present in lower amount (2220.0 µg/mL) in SN extracts from co-culture in comparison to CA produced by L. reuteri in monoculture (4687.9 µg/ mL), corresponding to a decrease of 52%.

Indeed, this is a logical correlation with esculetin findings, since the majority of esculetin comes from trans-CA, after subsequent transformation as we described previously (see Scheme 4). In fact, CA was the major metabolite after biotransformation, in both mono and co-cultures ( Figure 42). Moreover, we can observe on the chromatograms (see Figure 40), a significant decrease of the cis form of both CA and 5-CQA, in the co-culture biotransformation study. Nevertheless, we can not give a precise concentration because they are not available as analytical standards in their cis form. In the same line, with trans-CA, 5-CQA was less present in co-culture SN extracts when compared to those of monoculture. Finally, the yield of conversion of 5-CQA into metabolites was slightly higher in the co-culture (89.7%) than in the monoculture (83.6%). From a growth perspective, the presence and/or metabolites of bacteria do not seem to play a role in their growth. Furthermore, this study highlighted no detection of any new metabolite, which may come from trophic networking between bacteria. The increase of esculetin in co-culture could be explained by cross-feeding process, when somehow B. fragilis helped its formation. For example, it could contribute to the production of its intermediate, cis-CA, and its oxidised form, as this species possesses the same type of isomerase and oxidase found in L. reuteri. Another hypothesis could be that B. fragilis provide to L. reuteri a source of energy, which could help this last strain to increase its biotransformation ability.

Herein, we highlight a particular pattern of 5-CQA biotransformation between the two bacteria studied in co-cultures (B. fragilis and L. reuteri) in comparison to L. reuteri alone.

Interestingly, the formation of esculetin was higher in the co-culture biotransformation than when biotransformation was carried out only by L. reuteri. Contrary, trans-CA, and trans-5-CQA showed significant decreases in their amounts.

In perspective, we would like to conduct further studies using co-culture of the three bacteria studied (L. reuteri, B. fragilis and B. longum) to evaluate their potential impact on the formation of 5-CQA bioconverted metabolites. Besides the fact that bacteria metabolize polyphenols, one the other hand, they may modify the composition and function of gut bacteria.

For this purpose, we investigated the impact of5-CQA supplementation on the growth and metabolism of all strains studied.

II.C. Supplementation studies with 5-CQA

II.C.1. Polyphenols: in frontiers of prebiotics

Gut microbiota is involved in several functions of the human body. Among others, it provides essential nutrients and bioactive metabolites which are involved in host biology processes, including the production of (SCFAs), synthesis of vitamins and amino acids,

Hypothesis

Our study investigatedon the biotransformation of 5-CQA by three bacteria, L. reuteri, B. fragilis and B. longum highlighting its biotransformation into several metabolites only by L.

reuteri, but with a higher quantity when L. reuteri was co-cultured with B. fragilis. In addition, the well-established interaction between the gut microbiota and polyphenols also has a direct impact on the gut microbiota composition. Following our objective of investigating 5-CQA, the aim of this study was to evaluate its effects on the growth and metabolism of selected species such as L. reuteri and B. fragilis, which presented direct or indirect play on 5-CQA biotransformation. We aimed to provide more information on the potential role of 5-CQA, as a natural compound, in positively modulating the microbiota in favor of specific gut bacteria species that can ensure intestinal homeostasis and, in turn, favorably affect human well-being.

II.C.2. Results

II.C.2.1. Impacts of 5-CQA on the bacterial growth

In this study, the impact of different concentrations of 5-CQA, 0.1, 2.5, 50 and 1000 µM were evaluated on two intestinal bacterial strains L. reuteri and B. fragilis. This concentration range was selected from the lowest concentration to physiological ones that could potentially reach the intestine. 5-CQA was added at the beginning of the culture then bacteria were incubated inan anaerobic chamber for30 hours at 37°C. The culture liquids were collected at the beginning (time 0) and after 6, 24, and 30 hours of incubation to measure the OD600 nm reflecting bacterial growth and to determine the bacterial concentration (CFU.mL -1 ).

Cultures of the tested strains without 5-CQA were also performed as controls, referred to as corresponding to 6.56 x 10 8 CFU.mL -1 . After treatment using different 5-CQA concentrations, the growth curves of B. fragilis were still quite similar to controls, with a slight impact visualized at T6 h and T24 h. 5-CQA, at 2.5 µM, led to a slightly increase of B. fragilis growth, reaching values, at T6h, of 8.65 x 10 8 versus control sample. The same trend was observed at T24h, where B. fragilis have grew until 8.56 x 10 8 CFU.mL -1 in presence of 5-CQA (2.5 µM) and until 5.68 x 10 8 CFU.mL -1 in control. At T24h, we observed a bacterial growth reduction (around 14%) using 5-CQA at 50 µM compared to untreated bacteria. An increase of 51 % was noticed after treatment with 2.5 µM of 5-CQA. At T30h the effect of 5-CQA on the bacterial culture 123 disappeared to reach around the same value (5.46 x 10 8 CFU.mL -1 ) at different 5-CQA concentrations compared to untreated B. fragilis. The assay with 0.1 µM of 5-CQA added on B. fragilis culture must be considered incomplete, due to the poor number of bacteria at T6h, probably owing to a handling error.

Nevertheless, at T24h and T30h we noted a decrease (73% reduction) in the growth of B. fragilis in presence of 0.1 µM 5-of CQA with a value of 1.51 x 10 8 CFU.mL -1 and 1.45 x 10 8 CFU.mL - 1 compared to untreated bacteria (5.68 x 10 8 CFU.mL -1 at T24h and 5.46 x 10 8 CFU.mL -1 at T30h).

b) Effects of 5-CQA on L. reuteri growth

According to the results depicted in the Figure 44, untreated L. reuteri, in our experimental conditions, has exhibited a growth culture with a maximum reached at T30h corresponding to 6.61 x 10 7 CFU.mL -1 . Nevertheless, bacteria in the presence of 5-CQA has displayed a smaller exponential growth phase compared to untreated bacteria, with the maximum reached at T6h. The effect of 5-CQA was concentration-dependent (Figure 44)

although, not in a proportional correlation. In fact, 5-CQA at 0.1 µM and 2.5 µM had the most potent stimulation effect on growth of L. reuteri at T6h corresponding to 1.78 x 10 8 CFU.mL -1 (257% increasing) and 1.19 x 10 8 CFU mL -1 (138% increasing) respectively while the number of bacteria reached 4.99 x 10 7 CFU mL -1 for untreated samples. On the contrary, slightly effects

A) B)
were observed on L. reuteri growth with 50 µM and 1000 µM of 5-CQA, where the values reached 5.54 x 10 7 and 5.27 x 10 7 CFU.mL -1 , respectively. Moreover, the effectiveness of 5-CQA stimulation has been lost over time in each condition. L. reuteri culture at T30, reached a value of 2.40 x 10 7 CFU mL -1 under treatment with 0.1 µM of 5-CQA (corresponding to a 64 % reduction). A significant decrease compared to control (untreated L. reuteri) was observed after treatment with 1000 µM of 5-CQA. It is estimated to 84% of reduction, from 6.61 x 10 7 CFU mL -1 to 1.09 x 10 7 CFU.mL -1 . It was noted that 50 µM of 5-CQA was found to be ineffective, and 5-CQA at 1 mM did not stimulate the growth of L. reuteri. Therefore, it could be hypothesized that these concentrations are too high for bacteria, affecting adversely their growth. The GC-MS data obtained for L. reuteri samples were less reproducible than those for B. fragilis. Although these results must be analyzed with caution, a trend has be reported. A total of eight compounds have been identified by GC-MS analysis in all the L. reuteri samples at T0. These metabolites correspond to those found for B. fragilis, such as acetic acid (2), propanoic acid (3), butyrolactone (5), butyric acid (6) and isovaleric acid (7). Indeed, formic acid was detected, specifically produced by L. reuteri with or without 5-CQA supplementation. A variation in the production of some metabolites was noted, because L.

reuteri produced low levels of these metabolites. Two characteristics of L. reuteri metabolic profiles can be underlined independently of the supplementation: a) high levels of acetic acid L. reuteri a lactic acid bacterium produces lactic acid as a major organic acid. However, this metabolite was not detected in our study, since lactic acid is a non-volatile compound, and that cannot be quantified by GC-MS analysis. Lactic acid is known for its beneficial properties, mainly its immunomodulatory effect The L. reuteri strain was also able to produce formic acid in our conditions even if we observed non-reproducibility of its presence in all samples studied. This compound, systematically named methanoic acid, is the simplest carboxylic acid, with the chemical formula HCO2H. It is an important intermediate in chemical synthesis and occurs naturally, most notably in some ants. Indeed, the word "formic" comes from the Latin word for ant, formica, referring to its early isolation by the distillation of ant bodies. Moreover, it has been found that formic acid is able to reduce the growth of coliform and of the pathogenic Salmonella typhimurium GBL production might also be correlated with the production of other metabolites as it has been reported that A-factor, another GBL, auto-induces morphological differentiation and production of secondary metabolites in Streptomyces griseus [START_REF] Horinouchi | Hormonal control by A-factor of morphological development and secondary metabolism in Streptomyces[END_REF].

Moreover, 2-pyrrolidone, as the major metabolite of B. fragilis, is a precursor of gamma-aminobutyric acid (GABA), produced in the CNS as an inhibitory neurotransmitter [START_REF] Fasolato | Effect of 2-pyrrolidone on the concentration of GABA in rat tissues[END_REF]. In addition, pyrrolidone can cross cell membranes and BBB much more effectively than GABA, duo to its chemistry, as it is an uncharged molecule in physiological conditions [START_REF] Fasolato | Effect of 2-pyrrolidone on the concentration of GABA in rat tissues[END_REF]. Notably, a strain of B. fragilis was reported to produce a strong bioactive compound pyrrolopyrazine, which is known to exhibit antibacterial, antitumor, anti-angiogenesis and antioxidant activities [START_REF] Ser | Presence of antioxidative agent, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-in newly isolated Streptomyces mangrovisoli sp[END_REF]; nevertheless, this metabolite was not detected in our analysis.

(
In conclusion, we have underlined that 5-CQA had no effect on the metabolic production (in terms of SFCAs and BFCAs) of the two bacterial strains studied.

a) Impacts of 5-CQA supplementation in L. reuteri and B. fragilis strain as prebiotic

These in vitro investigations in vitro of growth the rate and patterns of L. reuteri and B.

fragilis under 5-CQA treatment showed that this natural compound can only influence the growth of L. reuteri.

After supplementation, 5-CQA at 0.1 µM and 2.5 µM has at T6h, the strongest stimulation effect on L. reuteri, the known probiotic strain. On the contrary, the growth of B.

fragilis was not stimulated by any 5-CQA concentrations, despite that, at T24h and T30h, we noted a slight decrease in its growth after treatment at 0.1 µM. These data highlighted that 5-CQA can potentially stimulate the L. reuteri growth at the lowest concentrations evaluated.

Overall, polyphenols have shown the ability to positively shape gut microbiota composition, and consequently could have positive impact on intestinal barrier integrity, and finally on human health. The mechanism by which polyphenols can exert their prebiotic effects is not fully elucidated. However, some modes of action have been proposed The prebiotic effects of polyphenols seem to be bacteria-dependent. In our study, we hypothesise that the effects of 5-CQA, only observed on L. reuteri, may be mediated through some PAZymes. As an example, only L. reuteri had shown, in our previous study, cinnamoyl esterase activity and capacity to biotransform 5-CQA. This hypothesis could be supported since by LC-MS/MS we have detected the same metabolites in this supplementation study than those detected in the biotransformation study (Figure 53). Moreover, this hypothesis is in line with findings from [START_REF] Li | Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers[END_REF]. They have shown that the pomegranate extract has promoted the growth of A. muciniphila only in participants for those their gut microbiota was able to produce urolithin-A, a gut-derived metabolites of ellagitannins and ellagic acid, the main polyphenols present in pomegranate [START_REF] Li | Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers[END_REF]. The prebiotic effect seems decreased when the 5-CQA concentrations increase. Nevertheless, this impact could not be correlated with the SCFAs and BCFAs production by both intestinal bacteria as there were not significantly affected by the 5-CQA supplementation. To try to get closer to the human intestine and to avoid use of animal models, Dr Latifa BOUSARGHIN and her team (NuMeCan), have developed an in vitro quadruple model (Figure 54). This original model is composed of enterocytes (Caco-2), mucus-secreting goblet cells (HT29-MTX), microfold cells (M cells) and enteroendocrine cells (NCI). Five cell types are used for the following, and therefore need to be cultured in specific conditions. The first types are NCI-H716 cells, floating cells mimicking enteroendocrine cells, obtained from a 33-yearold man with adenocarcinoma colorectal. Then Caco-2, mimicking enterocytes, are adherent cells from a 72-year-old man with of colorectal adenocarcinoma. The HT29-MTX line represents goblet cells, mucus producers obtained from HT29 colon cells of a 44-year-old patient with colorectal adenocarcinoma. The Raji-B cells, corresponding to floating cells, are lymphoblasts obtained from an 11-year-old boy. They allow for the differentiation of Caco-2 cells into M cells through mediators. Finally, the cells of type THP-1 are monocytes, floating cells, from a one-year-old child with acute monocytic leukemia.

Hypothesis

Given the importance of the intestinal barrier to health and considering the role of polyphenols in protecting its integrity and function, we aim to give information on the potential effects of 5-CQA and its gut-derived metabolites (CA and esculetin) on epithelial cells as a part of intestinal barrier integrity. Particular attention was paid to TEER, occludin and Zo-1 gene expression. In this study, we used the original in vitro quadruple model described previously.

II.E.2.

Results and discussion

Considering that 5-CQA reaches the colon at µM concentration and its microbial metabolites such as CA and esculetin could be close to the gut epithelium when produced; we have investigated whether these compounds could have a direct impact on epithelial cell function. To address these issues, we have used a quadruple in vitro model in the presence of above-mentioned polyphenols at 1 mM, 50 µM and 0.1 µM (from the lowest concentration to physiologically ones that potentially could reach the intestine) in basal condition. Different measurements will be evaluated in the presence of the tested polyphenols including cell viability, transepithelial electrical resistance (TEER) and gene expression such as occludin and Zo-1.

II.E.2.1. Cell viability in presence of polyphenols

Cell cytotoxicity was determined using the LDH method. This method is based on the To study the effects of 5-CQA, CA and esculetin on intestinal tight junctions, real-time PCR was performed and the expression of selected gene were evaluated.

a) Effects of polyphenols on occludin expression

The relative expression of occludin in the presence of 5-CQA, CA and esculetin at different concentrations compared to that in untreated cells, is shown in Figure 57. Occludin expression was increased by esculetin in a dose-dependent manner. Interestingly, the highest effect of esculetin (p < 0,001) was reached significantly at the lowest concentration, 2.5 µM, compared to the other treatmentsused. Significant results were also observed with esculetin treatment at 2.5 µM compared to those with CA (p < 0,01) and untreated cells (p < 0,05).

However, a slight increase was observed with esculetin treatment at 50 µM and 1 mM. In contrast, CQA and CA showed no significant effects on occludin expression, at these two concentrations. As none of the polyphenols tested were able to increase TEER, it is likely that the effects of esculetin on barrier integrity are due to the regulation of gene expression. Interestingly Zo-1 was differentially expressed in the presence of polyphenols compared to occludin. At 2.5 µM, only esculetin significantly induced Zo-1 expression compared to CA and 5-CQA (p < 0,05). However, higher effects were observed wen the concentration of esculetin was increased. This coumarin has increased significantly (p < 0,01), at 50 µM, zo-1 expression compared to untreated cells and CA, and toa lesser extent than to 5-CQA (p < 0,05). The highest effects of esculetin on zo-1 were observed a concentration of 1 mM, with p < 0,001 compared to 5-CQA and CA and p < 0,01 when compared to untreated cells.

Figure 58. Effects of polyphenols at 2.5 µM, 50 µM and 1 mM on relative expression of Zo-1 after incubation for 24 h

E.3.Conclusion

In our study, 5-CQA, CA and esculetin have exhibited no effect on TEER when evaluated using a quadruple model under basal conditions. Nevertheless, the role of esculetin, as a gut-derived metabolite of 5-CQA, was highlighted. This compound significantly increased the relative mRNA expression of tight junction proteins such as occludin and Zo-1. In contrast, 5-CQA and CA did not affect in the regulation of gene expression. These differences among the polyphenols studied suggest that their effects on the intestinal barrier are related to their molecular structures.

Further studies are ongoing, in an inflammatory model, to investigate the role of these selected polyphenols on gut barrier integrity and the immune response that occurs during intestinal inflammation. The data generated in vitro will give us insight into the potential effects of polyphenols and their gut-derived metabolites on gut-microbiota crosstalk, which could open new era of targeting the gut-brain axis to improve and even treat brain disorders.

Chapter III III. Conclusion and perspectives

A large body of research has indicated at profound role of polyphenols in human health to hydrolyse the ester bond between caffeic and quinic acids. Then, cis-CA is putatively formed by an isomerase present in the studied L. reuteri strain and, then it can be converted by an oxidase into caffeoyl-o-quinone. In the end, this latter is stabilized to esculetin. This coumarin was also identified after the electrochemical or enzymatic oxidations of CA, but in much smaller amounts compared to the biotransformed extracts. Indeed, these results provide insights into the biotransformation pathway of a natural compound, 5-CQA, which is highly ingested by the diet. Moreover, esculetin, is a molecule of great interest in medical research (Garg et al., 2020).

(
It has already shown some biological activities such as inhibition of oxidative stress and inflammation; it is also known to possess anticancer properties (Zhang et al., 2022). Some coumarins have been approved by the FDA as drugs, and warfarin is an example. It blocks vitamin K reductase and thereby disrupting the clotting mechanism. Much work is needed to bring these types of compounds, particularly esculetin, in nutraceutical forms to the clinical trial stage.

In addition, since there is interplay between bacteria in the gut, we studied whether such interactions could influence the biotransformation process. For this purpose, we have cocultured L. reuteri species that is involved in the biotransformation of 5-CQA with B. fragilis, which did not show the ability to biotransform it. As a preliminary result, esculetin formation was favorably increased by 23.9 % by co-culture of these two bacteria compared to L. reuteri alone. The next step will be to subject 5-CQA to the biotransformation process involving the three bacteria studied, L. reuteri. B. fragilis and B. longum.

Another question that needs to be addressed is how polyphenols and their gut-derived metabolites also modulate the composition and function of the gut microbiota, which has a positive impact on human health (Selma et al., 2020). Thus, we have studied the impact of 5-CQA on the growth and metabolic profiles of L. reuteri and B. fragilis. Interestingly, only L.

reuteri was affected by the presence of 5-CQA, with a positive correlation of growth at 0.1 and 2.5 µM. The mechanism by which 5-CQA have stimulated growth, could involve PAZymes, a fact of presence of this kind of enzymes was revealed in the biotransformation process.

Nevertheless, 5-CQA did not show any effect on metabolism in either L. reuteri or B. fragilis.

On the other hand, maintaining the homeostasis in the gut or even more, restoring it, could be the key to alleviate and treat brain disorders. In this research context, we have investigated the role of 5-CQA and its gut-derived metabolites, CA and esculetin, on barrier function by using an in vitro quadruple model. Interestingly, esculetin has increased the relative mRNA expression of tight junction proteins such as occludin and Zo-1. Our study supports the evidence of the role of this coumarin as a natural compound to positively impact their intestinal barrier. Nevertheless, neither CA and nor CQA, exhibited effects on the expression of these selected genes. These data are in basal condition, and further studies are underway, using an inflammatory model, with the aim of providing more information on the potential effects of these phenolic compounds on inflammatory pathways that may be involved in the gut-brain axis crosstalk.

From a general point of view, it is clear, that polyphenols can modulate the gut microbiota and consequently impact host health, making them a promising approach to improve brain Bacteria were grown in BHI broth under anaerobic conditions using an anaerobic chamber (Macs-VA500, Don Whitley) flooded with 80% N2, 10% H2 and 10% CO2). Kinetics of cultures were monitored by measuring absorbance (optical density (OD) at 600 nm) and colony-forming units (CFU.mL -1 ). The cultures of bacteria (150 mL) were established by suspending the fresh bacterial strains isolation colonies (from a 48h culture in anaerobic conditions at 37°C) in BHI standard medium in order to obtain an appropriate inoculum of each bacterium; for L. reuteri 10 7 CFU.mL -1 while for B. fragilis and B. longum 10 8 CFU.mL -1 .

Then, cultures and a flask containing only BHI standard used as a control of the purity, were incubated in anaerobic chamber for 30 h.

IV.B. Electrochemical Procedures

Before each measurement, a 2 mM 5-CQA or CA solution was freshly prepared in 0.1 M PBS pH 5.8 and nitrogen saturated during 5 min at room temperature. All potentials reported herein are vs. Ag/ AgCl reference electrode.

IV.B.1. Cyclic voltammetry (CV)

CV experiments were carried out on a dual potentio-galvanostat PGSTAT100 (Autolab instrument, Eco Chemie B.V., Utrecht, The Netherlands). Measurements were performed with a three-electrode cell at room temperature. A glassy carbon disk working electrode (Ø2 mm), a platinum wire counter electrode and a reference electrode, Ag/AgCl/KCl 3 M, were used.

Before each measurement, the glassy carbon disk working electrode was polished on a waterproof Silicon Carbide Paper 4000 (Struers) using a 0.3 µm alumina suspension, washed with distilled water and dried. The CV were recorded at a scan rate 0.1 V.s -1 under nitrogen atmosphere. Epa and Epc are relative to anodic and cathodic potential, respectively; ia and ic to anodic and cathodic current respectively.

IV.B.2. Spectroelectrochemical measurements

Spectroelectrochemical studies were conducted using a combined potentiostat/ galvanostat (AUTOLAB PGSTAT 204, Metrohm, Switzerland) and spectrometer (Avantes, AvaSpec-ULS20 48) equipped with a UV-Visible deuterium/ halogen light source (200 -2500 nm). This system was controlled by NOVA 1.11 software. A three-electrode cell was used comprising a platinum grid working electrode (7 x 6 mm, wire diameter 0.3 mm), a platinum wire counter electrode and an Ag/ AgCl/ KCl 3M reference electrode placed in a spectrophotometric quartz cuvette (SEC-C thin layer quartz cristal spectroelectrochemical cell, 1 mm optical path length (ALS CO., Ltd)). Before each measurement, the platinum grid working electrode was washed with H2SO4 0.2 M and sonicated for 5 minutes. Then, a cleaning was done by recording 200 cyclic voltammograms in H2SO4 0.2 M between -0.5 V and + 1.5

V (scan rate 0.5 V.s -1 ). In a second step, the platinum grid was cleaned recording 200 cyclic voltammograms in PBS pH 5.8 between -1.2 and + 1.5 V (scan rate 0.5 V.s -1 ).

III.B.3. Linear Sweep Voltammetry (LSV)

The LSV (i = f (E)) were firstly recorded at a scan rate of 0.005 V.s-1. UV-Vis spectra were simultaneously recorded between 200 and 600 nm.

III.B.4. Electrolysis

Electrolysis were performed by a chronoamperometry technique i = f (t) at a fixed potential during 1200 s for 5-CQA and CA. The applied potential was fixed at + 0.8 V for the 5-CQA electrolysiS and + 0.6 V for CA The UV-Vis spectra were simultaneously recorded.

During electrolysis, nitrogen bubbling was maintained, first to avoid any oxidation derived from oxygen and also to mix the solution (the whole cell volume is 600 µL, whereas the optic way compartment holds approximately 200 µL of the solution). After electrolysis, the whole solution was retrieved, saturated with argon in a sealed vial and stored at -20°C.

IV.C. Enzymatic procedures by polyphenol oxidase (PPO)

The procedure were adapted from Le [START_REF] Bourvellec | Inhibition of Apple Polyphenol Oxidase Activity by Procyanidins and Polyphenol Oxidation Products[END_REF] and Cheynier et al. (Cheynier and Moutounet, 1992b) 4.5 ml of 5-CQA or CA 5 mM dissolved in malate buffer 30 mM at pH 4.8 reacted with 500 µL polyphenol oxidase (PPO) malate buffer solution. The whole reaction was stirred during 5 sec then filtered on PVDF 0.45 µm just before LC-DAD-ESI-MS 2 analysis.

IV.C. Biotransformation study

IV.C.1. Biotransformation method

Resting cells biotransformation studies were carried out when bacteria reached stationary phase (at 30 h of growth), by adding solution of 5-CQA (1 mM) in PBS (50 mL; pH 5.8) on biomass (pellet) of each bacterial strain separated from the culture medium by centrifugation (3500 rpm, 4°C, 15 min). In parallel, as controls (50 mL, pH 5.8), PBS alone or with 5-CQA, CA (1 mM) were incubated without bacteria. In addition, two more controls were prepared either incubating bacteria in BHI or in PBS. Other biotransformation experiments were performed using the same condition, except BHI instead of PBS at 5.8. They were incubated at 37°C, 120 rpm under anaerobic condition using sealed jars and anaerobic bags.

After 24 h of incubation, the samples were centrifugated at 3500 rpm, at 4°C for 15 min. The extraction process was further performed on pellet and on supernatant (SN) using ethyl acetate

(2 x 10 mL for pellet and 2 x 25 mL for SN).

In the detection mass was performed with a noise level at 1.10 5 for MS 1 and 1 for MS 2 Molecular features were detected using the centroid algorithm followed by a chromatogram building step.

The chromatograms were built with the ADAP algorithm with a minimum group size of 2, a group intensity threshold of 1.10 5 , a minimum highest intensity at 1. and an m/z tolerance at 0.8 m/z. The ADAP wavelet algorithm was then used for deconvolution with the following settings: S/N threshold: 10, S/N estimator: Intensity window SN, min feature height: 200, coefficient/area threshold: 10, peak duration range: 0.01-10, RT wavelet range: 0.-0.50. The isotopic peak grouper algorithm was then used with an m/z tolerance at 0.8 m/z, an RT tolerance at 0.5 min and a maximum charge of 2. The peaks were aligned in a unique peak list using the join aligner algorithm with the following parameters: m/z tolerance: 0.8, weight for m/z: 1, retention time tolerance: 0.5 min, weight for RT: 1, require same charge state: unchecked, require same ID: unchecked. Finally, the peak list was filtered using the peak list rows filter by keeping only the peaks with MS 2 scan (GNPS). The peak list was exported to a MGF file and a csv file gathering the metadata. The MGF file was imported using the Python v3.7 library matchms v0.9.2, a MN was generated and dereplicated using the GNPS spectral libraries.

Clustering threshold was set at 0.6, and the dereplication parameters were the following: cosine The colonies are then counted in the dishes in order to enumerate the viable and cultivable bacteria. For the control of purity, the dishes were prepared by spreading bacteria from every culture in each time.

Then, at each stage, 5 mL of each sampling time was taken and then centrifuged for 15 minutes at 3500 rpm in 4° C. Thereafter, the supernatant separated from the pellet, was used to well plate was incubated in the dark during 30 min at ambient temperature. After incubation the well lecture was performed using a spectrophotometer AMR-100 Microplate reader using 492 nm wavelength which is corresponded to the red formazan chromogen phenomenon observation. Value absorbances have been subtracted to those of controls (apical middle without cells).

III.E.6. Expression level of selected genes by quantitative reverse transcription PCR (RT-qPCR)

After 21 days of co-culture, RNA was extracted from the upper chamber using Total RNA and Protein isolation kit (Macherey-Nagel) according to the manufacturer's instructions.

Afterward, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to reverse-transcribe the RNA into cDNA. Then, the genes that were selected specifically for each cells,were relatively quantified using StepOnePlus (Applied Biosystems) with the SYBR Green PCR Master Mix (Applied Biosystems) (Le [START_REF] Bars | Impact of a mutator phenotype on motility and cell adherence in Salmonella Heidelberg[END_REF]. To investigate genes of tight-junction components, we have selected occludin and Zonula occludens (Zo-1) genes.

Each gene was normalized to the TBP (TATA box binding protein) mRNA expression level before calculating the fold-change values. Relative gene expression was calculated by the 2 -ΔΔCT method (Livak and Schmittgen, 2001).

III.E.7. Statistical Analysis

All experiments were performed at least in triplicates. After normality of data, distribution was confirmed using the Shapiro-Wilk test, differences within and between groups were calculated using a two-way analysis of variance (ANOVA). Data presented as mean ± SD and p-value less than 0.05 was considered as significant.

Résumé :

Les polyphénols sont connus pour leurs propriétés antioxydantes et comme modulateurs des voies de signalisation redox, ce qui a un effet positif sur la santé humaine. Compte tenu de leur supériorité en termes de présence dans la circulation systémique et de leur bioactivité souvent plus élevée que celle des polyphénols parents, on s'intéresse aujourd'hui davantage au rôle central du microbiote intestinal dans la production de métabolites de faible poids moléculaire. Ainsi, nous avons concentré notre étude sur l'acide 5-O-caféoylquinique (5-CQA), un polyphénol alimentaire, soumis à une étude de biotransformation en utilisant Lactobacillus reuteri, Bacteroides fragilis et Bifidobacterium longum. Notre étude a mis en évidence la capacité de L. reuteri à bioconvertir le 5-CQA en plusieurs métabolites, et notamment en un composé naturel connu, l'esculétine. Cette capacité de biotransformation a été également évaluée en co-culture. Cette étude a mis en lumière, pour la première fois, une voie oxydative intéressante réalisée par les bactéries intestinales. Des études électrochimiques et enzymatiques ont permis d'identifier, après analyse par LC-MS/MS, les composés oxydés du 5-CQA et de l'acide caféique. De plus, l'esculétine a montré un effet bénéfique sur l'intégrité de la barrière épithéliale par une étude originale réalisée in vitro sur un modèle intestinal constitué de quatre types de cellules.

Title: 5-O-caffeoylquinic acid, a dietary polyphenol, and its gut bacteria-derived metabolites: electrochemical, LC-MS/MS, biotransformation and biological studies Keywords: polyphenol, gut microbiota, biotransformation, LC-MS/MS, electrochemistry, gut barrier integrity

Abstract:

Polyphenols are known for their antioxidant properties and as modulators of redox signalling pathways, which have a positive effect on human health. Given their superiority in terms of presence in systemic circulation and their often higher bioactivity than parent polyphenols, there is now more interest in the gut microbiota`s pivotal role in the production of low molecular weight metabolites. Thus, we have focused our study on 5-O-caffeoylquinic acid (5-CQA), a dietary polyphenol, subjected to a resting cell biotransformation study using Lactobacillus reuteri, Bacteroides fragilis and Bifidobacterium longum. Our study highlighted the ability of only L. reuteri to bioconverse 5-CQA into several metabolites, including a known natural compound esculetin. This biotransformation capacity was also evaluated in co-culture. This study put in the spotlight, for the first time, an interesting oxidative pathway carried out by gut bacteria. Electrochemical and enzymatic studies led to identify, after LC-MS/MS analysis, the oxidized compounds of 5-CQA and caffeic acid. In addition, esculetin has shown a beneficial effect on the integrity of the epithelial barrier using an original in vitro quadruple intestinal model.

III

  II.A.1.3. Electrochemistry ............................................................................................. II.A.2. Results and discussion .......................................................................................... II.A.2.1. Cyclic voltammetry ........................................................................................ II.A.2.2. Spectroelectrochemical analysis of CQA and CA ......................................... II.A.2.3. LC-MS 2 analysis of electrochemical and enzymatic oxidations of 5-CQA and CA ................................................................................................................................. a) Characterization of oxidized products of 5-CQA after electrochemical and enzymatical oxidation ............................................................................................................................... b) Characterization of oxidized products of CA after electrochemical and enzymatical oxidations .............................................................................................................................. II. B. Biotransformation of 5-CQA by gut bacteria in mono and co-culture ............... II.B.1. The characteristics and properties of three studied gut bacteria ........................... II.B.1.1. Taxonomy and ecology .................................................................................. a) Lactobacillus reuteri ...................................................................................................... b) Bacteroides fragilis ....................................................................................................... c) Bifidobacterium longum ............................................................................................... II.B.1.2. The roles in intestinal health of L. reuteri, B. fragilis and B. longum ............ II.B.2. Biotransformation of 5-CQA by L. reuteri, B. fragilis and B. longum ................. II.B.2. 1. Introduction ................................................................................................... II.B.2. 2. Results and discussion ................................................................................... a) Biotransformation studies of 5-CQA ................................................................................ b) Characterization of oxidized compounds of 5-CQA and CA by electrochemical and enzymatic methods .............................................................................................................. b-1) Electrochemical studies of 5-CQA and CA ................................................................. b-2) Comparison of LC-DAD-ESI-MS 2 of biotransformed, electrolyzed and enzymatic samples. ............................................................................................................................... II.B.2.3. Conclusions .................................................................................................. II.B.3. Biotransformation of 5-CQA by co-culturing in vitro two gut bacteria, L. reuteri and B. fragilis ................................................................................................................. II.B.3.1. Context of the study ...................................................................................... II.B.3.1. Results and discussion .................................................................................. a) Growth kinetics of L. reuteri and B. fragilis in co-culture .............................................. b) Impact of bacterial interactions on the biotransformation of 5-CQA ............................. II.C. Supplementation studies with 5-CQA ................................................................... IV II.C.1. Polyphenols: in frontiers of prebiotics ................................................................ II.C.2. Results ................................................................................................................. II.C.2.1. Impacts of 5-CQA on the bacterial growth .................................................. a) Effects of 5-CQA on B. fragilis growth .......................................................................... b) Effects of 5-CQA on L. reuteri growth ........................................................................... II.D.2.2. Effects of 5-CQA on metabolic production of B. fragilis and L. reuteri ..... a) Effects on SCFAs and BCFAs production by B. fragilis ................................................ b) Effects of 5-CQA on SCFAs and BCFAs production by L. reuteri ................................ II.3. Discussion ............................................................................................................... II.3.1. Metabolic profiles of B. fragilis and L. reuteri strains ..................................... a) Impacts of 5-CQA supplementation in L. reuteri and B. fragilis strain as prebiotic ....... II.D.4. Conclusion .......................................................................................................... II.E. Biological study of 5-CQA and its gut-derived metabolites by quadruple original in vitro cell model .............................................................................................................. II.E.1. Roles of polyphenols on intestinal barrier ........................................................... II.E.2. Results and discussion ......................................................................................... II.E.2.1. Cell viability in presence of polyphenols ..................................................... II.E.2.2. TEER evolution in presence of polyphenols ................................................ II.2.3. Effects of polyphenols on gene expression ...................................................... a) Effects of polyphenols on occludin expression ............................................................... b) Effects of polyphenols on Zo-1 expression .................................................................... E.3.Conclusion................................................................................................................ Chapter III ............................................................................................................................ III. Conclusion and perspectives ..................................................................................... Chapter IV ............................................................................................................................ IV. Materials and methods .............................................................................................. IV.A. Generalities ........................................................................................................... IV.A.1. Reagents and chemicals ................................................................................. IV.A.2. Bacterial strains............................................................................................. IV.A.3. Growth conditions of L. reuteri, B. fragilis and B. longum ............................... IV.B. Electrochemical Procedures .................................................................................. IV.B.1. Cyclic voltammetry (CV) .................................................................................. V IV.B.2. Spectroelectrochemical measurements .............................................................. III.B.3. Linear Sweep Voltammetry (LSV) .................................................................... III.B.4. Electrolysis ......................................................................................................... IV.C. Enzymatic procedures by polyphenol oxidase (PPO) ......................................... IV.C. Biotransformation study ....................................................................................... IV.C.1. Biotransformation method ................................................................................. IV.C.2. In silico search of cinnamoyl esterase activity .................................................. IV.C.3. UV irradiation of samples .................................................................................. IV.C.4. LC-DAD-ESI-MS 2 analysis............................................................................... IV.C.5. Mzmine and GNPS data treatment .................................................................... IV.C.6. Determination of calibration curves for 5-CQA, CA, esculetin by LC-MS/MS ........................................................................................................................................ IV.D.2. Analysis of SCFAs, BCFAs, ............................................................................. IV.D.2.1. Extraction methods ..................................................................................... IV.D.2.2. Gas chromatography-mass spectrometry (GC-MS) analysis to determine SCFAs or BCFAs production ..................................................................................... IV.E. Intestinal cell study ................................................................................................ IV.E.1. Cell lines and growth culture ............................................................................. IV.E.2. Cellular model composed of four cells (quadricellular model) ......................... IV.E.3. Evaluation of the activity of esculetin, 5-CQA and CA on the quadricellular model .............................................................................................................................. IV.E.4. Transepithelial electrical resistance (TEER) measurements and paracellular permeability study .......................................................................................................... III.E.5. Cytotoxicity analysis by LDH (Lactate Deshydrogenase) release ..................... III.E.6. Expression level of selected genes by quantitative reverse transcription PCR (RT-qPCR)...................................................................................................................... III.E.7. Statistical Analysis .............................................................................................

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Representation of a phenolic structure .................................................................................... 6 Figure 2. Structures of main dietary polyphenols (Manach et al., 2004) ................................................ 9 Figure 3. General structures of chlorogenic acids (CGAs) and its chemical variations. CQA, caffeoylquinic acid; diCQA, dicaffeoylquinic acid; p-CoQA, p-coumaroylquinic acid; FQA, feruloylquinic acid; CFQA, caffeoylferuloylquinic acid ...................................................................... 15 Figure 4. Chemical structures of chlorogenic acid stereoisomers of 5-CQA: A) 1-CQA; B) 3-CQA; C) 4-CQA; D) 5-CQA (Lu et al., 2020). .................................................................................................... 18

  Figure 7. A schematic covering the microbiome nomenclature, highlighting the microbiota and their "theatre of activity" (Berg et al., 2020) ................................................................................................. 24 Figure 8. Consequences of low abundance of Bifidobacteria in infants (Korpela, 2021) ................... 27 Figure 9. Bioactivation of sulfasalazine by intestinal bacterial metabolism ......................................... 33 Figure 10. GALT organization of colon (Cader and Kaser, 2013). .................................................... 36

52 Figure 17 .Figure 24 .VIIIFigure 34 .Figure 52 .Figure 58 .

 521724345258 Figure11. The Network between gut bacteria and innate and adaptive immune system[START_REF] Sansonetti | The innate signaling of dangers and the dangers of innate signaling[END_REF] ..................................................................................................................................................... 37 Figure 12. Main metabolic functions of gut microbiota, including biotransformation of dietary ingested compounds, modification of host metabolites and de novo synthesis of metabolites (Silpe and Balskus, 2021) ..................................................................................................................................................... 38 Figure 13 .Gut microbial formations pathways of acetate, propionate and butyrate formations from dietary polysaccharide (Koh et al., 2016) ; abbreviations: PEP (phosphoenolpyruvate); DHAP, (dihydroxyacetonephosphate). .............................................................................................................. 39 Figure 14. The journey of SCFAs in humans, including production, absorption, metabolism, and distribution (Dalile et al., 2019) ............................................................................................................ 40 Figure 15. The role of SCFAs in human physiology function (Dalile et al., 2019). ............................. 42 Figure 16. The illustration of gut microbiota biotransformation reactions of some of the polyphenols including: Flavanones (Hesperidin;Citrus), isoflavones (Daidzin;soy); lignans (secoisolariciresinol diglucoside; flaxseed); ellagitannins (hexahydroxydiphenic glucoside; nuts and

  metabolic and neurodegenerative diseases (Carregosa et al., 2019a; Cortés-Martín et al., 2020). Indeed, a better understanding of the polyphenols-microbiota interplay will allow the development of novel nutraceutical strategies to prevent, and even more, alleviate various diseases. Considering that gut-derived metabolites have already shown interesting neuroprotective properties, we will do a focus on their impact on brain diseases. This Ph.D. work was carried out in the Natural Products group of COrInt (Chimie Organique aux Interfaces) team, whose main research activities concern lichens and their microflora as an original source of secondary metabolites and how these microorganisms can interact with their host. For my Ph.D. thesis, we have started a new topic thanks to the collaboration with one of the groups of NuMeCan team (Nutrition, metabolismes et Cancer), with the final objective of studying how diet can impact health and potentially prevent chronic disease.

1 .

 1 Chemistry and dietary sourcesPolyphenols are secondary metabolites, also known as 'specialized metabolites', found in the plant kingdom[START_REF] Chikezie | Bioactive Principles from Medicinal Plants[END_REF]. Notably, they are involved in plant defence, including protection against oxidative stress and ultraviolet radiation. In addition, they act as signalling compounds, that attract pollinators or animals for seed dispersal. They also contribute to the organoleptic characteristics of flowers, leaves, fruits and vegetables, such as bitterness, astringency, color and flavor (Naczk and Shahidi, 2004).In addition to their beneficial effects on plants, these compounds are of great interest because of their impact on human health. Epidemiological, clinical and nutritional studies strongly support the suggestion that polyphenols consumption is associated with the prevention of many major chronic diseases such as cardiovascular, neurodegenerative and cancer diseases (Del Bo' et al., 2019; Fraga et al., 2019; Pandey and Rizvi, 2009; Scalbert et al., 2005b).

Figure 2 )

 2 Figure 2) (Crozier et al., 2009).

Figure 1 .

 1 Figure 1. Representation of a phenolic structure

  Based on their chemical structures, they can be classified into two classes: flavonoids and non-flavonoid phenolic compounds, which are subdivided into different subgroups. The structures of the main flavonoid and non-flavonoid compounds are shown in Fig 2.

Figure 2 .

 2 Figure 2. Structures of main dietary polyphenols (adapted from[START_REF] Manach | Polyphenols: food sources and bioavailability[END_REF]

  acid, 4-O-caffeoylquinic acid and proanthocyanidin polymers, were the three most consumed individual polyphenols (Zamora-Ros et al., 2016).

I.A. 2 . 1 .

 21 Oxidative stress: Risk on health and possible fights Free radicals are defined by the presence of one or more unpaired electrons in their atomic or molecular orbitals making these molecules highly reactive (Valko et al., 2007). These molecules, known as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are produced during cellular respiration and normal metabolism (Phaniendra et al., 2015). The species mainly include superoxide anion free radicals (𝑶 𝟐 • -), hydroxyl free radicals (𝑯𝑶 • ), peroxyl radicals (𝑹𝑶𝑶 • ), nitrogen peroxide ( • NO) and others (Bergamini et al., 2004).

  oxidative stress can contribute to and trigger diseases. The production of reactive species during oxidative stress can directly oxidize macromolecules, leading to damage of cell function and death; second, it can cause misfolding of redox signalling (Forman and Zhang, 2021). In this context, polyphenols, have shown their ability to scavenge free radicals species and positively modulate redox signalling pathways, thereby contributing to reduced oxidative stress[START_REF] Gebicki | Fast Antioxidant Reaction of Polyphenols and Their Metabolites[END_REF].

  Zhang and Tsao, 2016). Many polyphenols have been reported to reduce oxidative stress through the induction of Nrf2-dependent gene expression. For example, quercetin has shown a strong neuroprotective effect by repressing NF-κB. Likewise, catechin, increased cell survival by downregulating the NF-κB and MAP kinase pathways (Di Meo et al., 2020). The list of polyphenols as modulators of signalling pathways activated in neurodegenerative diseases also includes resveratrol, curcumin and epigallocatechin-3-gallate apigenin, kaempferol, genistein (Jantan et al., 2021).

Figure 3 .

 3 Figure 3. General structures of chlorogenic acids (CGAs) and its chemical variations. CQA, caffeoylquinic acid; diCQA, dicaffeoylquinic acid; p-CoQA, p-coumaroylquinic acid; FQA, feruloylquinic acid; CFQA, caffeoylferuloylquinic acid

Figure 4 .

 4 Figure 4. Chemical structures of chlorogenic acid stereoisomers of 5-CQA: A) 1-CQA; B) 3-CQA; C) 4-CQA; D) 5-CQA (Lu et al., 2020)

  Figure 5 (Tomas-Barberan et al., 2014). There are two proposed colonic biotransformation pathways for QA (Figure 5): (1) a reductive route leading to cyclohexane carboxylic acid, and (2) an oxidative route leading to aromatic compounds (Pinta et al., 2018). However, the pharmacokinetic properties of chlorogenic acids have not been fully elucidated and further studies are encouraged to be undertaken (Clifford et al., 2020).

Figure 5 .

 5 Figure5. Representative scheme of absorption and metabolism pathway of 5-CQA in humans (I) almost one-third of 5-CQA intake is absorbed in upper digestive (stomach and/or small intestine, without hydrolysis and then enter to the circulatory system, (II) a small amount of the 5-CQA (around 7%) is hydrolysed into CA and QA and then absorbed via the small intestine. (III) the major part reaches the colon and is metabolised by gut bacteria; (IV) 5-CQA as native form and its metabolites are absorbed and/or are subjected to liver metabolism, including methylation, by COMT = catechol-O-methyltransferase (COMT), glucuronidation (GT) or sulfation (ST),[START_REF] Lu | Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action, and future directions[END_REF]

Figure 6 .

 6 Figure6. An overview of the biological activities of 5-CQA in humans[START_REF] Lu | Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action, and future directions[END_REF]

(

  Korpela and de Vos, 2018). Moreover, C-section born infants are predisposed to future diseases. As an illustration, scientific evidence has revealed that Bifidobacteria are the most negatively affected gut bacteria in early life, mainly by C-section birth, antibiotic exposure and formula feeding. Thus, early low abundance of this bacteria is associated with a higher risk of developing immune and metabolic diseases in the coming years Figure8(Korpela, 2021).

Figure 8 .

 8 Figure 8. Consequences of low abundance of Bifidobacteria in infants[START_REF] Korpela | Impact of Delivery Mode on Infant Gut Microbiota[END_REF]

Figure 9 .

 9 Figure 9. Bioactivation of sulfasalazine by intestinal bacterial metabolism

Figure 10 .

 10 Figure10. GALT organization of colon[START_REF] Cader | Recent advances in inflammatory bowel disease: mucosal immune cells in intestinal inflammation[END_REF].

Figure 11 .

 11 Figure11. The Network between gut bacteria and innate and adaptive immune system[START_REF] Sansonetti | The innate signaling of dangers and the dangers of innate signaling[END_REF]

Figure 12 .

 12 Figure12. Main metabolic functions of gut microbiota, including biotransformation of dietary ingested compounds, modification of host metabolites and de novo synthesis of metabolites[START_REF] Silpe | Deciphering Human Microbiota-Host Chemical Interactions[END_REF]

Figure 13 .

 13 Figure 13. Gut microbial formations pathways of acetate, propionate and butyrate formations from dietary polysaccharide (Koh et al., 2016) ; abbreviations: PEP (phosphoenolpyruvate); DHAP, (dihydroxyacetonephosphate).

Figure 14 .

 14 Figure 14. The journey of SCFAs in humans, including production, absorption, metabolism, and distribution (Dalile et al., 2019)

Figure 15 .

 15 Figure15. The role of SCFAs in human physiology function(Dalile et al., 2019)

( 1 .

 1 [START_REF] Cryan | Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour[END_REF]. However, since they are unlikely to cross BBB, they do not affect brain directly. The mechanism by which these neuroactive substances produced in the gut affect the brain is unknown. As a hypothesis, they may act in the enteric nervous system (Sun et al., 2020). I.B.4. Gut microbiota in health and diseases Understanding the profound role of microbiota in health and diseases, has become one the most intense and explosion topic in the past decades (Fan and Pedersen, 2021; Lynch and Pedersen, 2016; B. Wang et al., 2017). Indeed, studies have shown there is a mutually beneficial relationship between microorganisms reside with the host (O'Hara and Shanahan, 2006). It is well established that the gut microbiota is involved into numerous physiological processes of the human body. including digestion, protection, immune maturation and function, brain development and function, as well. Moreover it contributes to our well-being and mostly positively influence our health (Adak and Khan, 2019). In turn, the host provides major sources of carbon and energy for gut bacteria (Bäckhed et al., 2005). In addition, altered gut microbiota composition and function has been linked with several metabolic and neuronal diseases (Yinwei Chen et al., 2021).(Wang et al., 2017). I.B.5. Gut microbiota linked to the brain disorders A large number of studies explained how gut dysbiosis can affect the brain and play a key role on the pathogenesis of neurodegenerative diseases (Schächtle and Rosshart, 2021; Shabbir et al., 2021). Moreover, an altered balance of gut microbiota has been linked to a range of brain disorders including Stress and Depression, Autism Spectrum disorders, Multiple Sclerosis (MS), Parkinson's disease (PD) and Alzheimer's disease (AD) (Vijay and Valdes, 2021; Zhu et al., 2020). Intriguingly, scientists detected differences of the gut microbiota profiles in patients with brain-related disorders compared to healthy controls (Fung et al.Anxiety and Major depressive disorders (MDD) Major depressive disorders (MDD) affect at least 4.4% of the world's population (Flux and Lowry, 2019). This disease is associated with the dysfunction of 5-hydroxytryptamine (5-HT, serotonin) and/or norepinephrine at specific synapses in the brain (Foster et al., 2021). Recently, numerous studies are supporting the concept that the gut microbiota is implicated in depression. For instance, several experimental in-vivo studies in animal models provide evidence for the involvement of the gut microbiota in stress-related disorders including anxiety and depression (Dinan and Cryan, 2013; Foster and McVey Neufeld, 2013). Interestingly, when the scientists transplanted fecal microbiota from MDD patients to germ-free rat model, they observed that the host began to exhibit depressive features such as anhedonia and anxiety-like behaviors, as well as an alteration in tryptophan metabolism (which is in humans linked to a variety of neuropsychiatric diseases) by an increase of kynurenine/tryptophan ratio (Kelly et al., 2016). In the same way, Zheng et al., showed that a lack of microbiota induced depression-like behavior in germ-free (GF) mice (Zheng et al., 2016). However, further studies shed the light on how the gut microbiota is linked to brain diseases. Those studies identified differences in the gut microbial composition of patients with anxiety and depression compared to controls (Aizawa et al., 2016; Jiang et al., 2015; Lin et al., 2017; Naseribafrouei et al., 2014). Anxiety has been characterized with overabundance of Escherichia, Shigella, Fusobacterium and Ruminococcus gnavus (Jiang et al., 2018), whereas Prevotella and Klebsiella were significantly increased in the fecal microbiota of patients with major depressive disorders (Lin et al., 2017). A large study from Flemish Gut Flora Project has shown further insights into the link between gut microbes and depression.

  Figure 16) (Espín et al., 2017). Subsequently, these biotransformation products undergo the Phase I and Phase II biotransformation and enter in the enterohepatic circulation, then reach tissues and brain where they exert their biological effects (Marhuenda-Muñoz et al., 2019).

Figure 16 .

 16 Figure 16. The illustration of gut microbiota biotransformation reactions of some of the polyphenols including: Flavanones (Hesperidin;Citrus), isoflavones (Daidzin;soy); lignans (secoisolariciresinol diglucoside; flaxseed); ellagitannins (hexahydroxydiphenic glucoside; nuts and berries); phenylpropanoids (chlorogenic acid; coffee). A) in their native form; B: Hydrolysis reactions mediated by gut microbiota; C: Ring cleavage and demethylation; D: Reduction reactions (dehydroxylation and hydrogenation) ( Espín et al., 2017)

Figure 17 .

 17 Figure 17. Structures of phenylacetic acids and phenylpropionic acids, evaluated for their neuroprotective effects

Figure 18 .

 18 Figure18. Structures of benzene diols and triols, evaluated for their neuroprotective effects on microglia cells.

Figure 19 .

 19 Figure19. Illustration of the polyphenols, parents compounds; isoflavones (Genistein and daidzein) and lignan (secoisolariciresinol) and their gut-derived metabolites (equol and enterodiol)(Johnson et al., 2019).

(

  Azad et al., 2018). The increased abundance of Roseburia, known as butyrate producing bacteria, led to the maintenance of gut health and immune function as well (Tamanai-Shacoori et al., 2017). Akkermansia muciniphila is specialized in mucin degradation, making this bacterium an important contributor to maintain intestinal mucosal barrier function (Zhang et al., 2019; Zhou and Zhang, 2019). In the same way, Faecalibacterium prausnitzii, known as immunomodulatory bacteria, inhibits inflammation by modulating inflammatory cytokines and interleukins (He et al., 2021; Touch et al., 2022). Furthermore, the promoting of commensal bacteria in turn provides suppression of the growth of opportunistic bacteria, leading to the maintain of the gut homeostasis (Y. Guo et al., 2020). I.C.2.2. Polyphenols acting as antimicrobial agents Another means to regulate gut microbiota composition is to limit the number of pathogenic bacteria and polyphenols have been extensively studied as antimicrobial agents against several known pathogenic bacteria (Marín et al., 2015). Escherichia coli, Helicobacter pylori, Klebsiella pneumonia, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa were significantly inhibited by polyphenols. A human study demonstrated that curcumin and turmeric decreased Blautia spp. and Ruminococcus spp. in the gastrointestinal tract (Peterson et al., 2018). Han et al. demonstrated that the strawberry decreased the levels of opportunistic bacteria such are Dorea and Bilophila, in a colitis induced model (Han et al., 2019). Enterococcus caccae was drastically inhibited by a flavanone naringenin treatment (Firrman et al., 2018). In another example, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus were inhibited by anthocyanins, flavan-3ols, B-type proanthocyanidins, and ellagitannins, as phenolic extracts obtained from eight berries (Kawabata et al., 2019). A more recent systematic review gathered all the evidence on the antimicrobial activity of different classes of plant polyphenols against resistant and nonresistant Gram-positive bacteria. Scientists concluded that the significant antimicrobial effect of both, polyphenols and mixtures, was achieved at low µg/mL range of Minimal Inhibitory Concentration (MIC) values (Álvarez-Martínez et al., 2020). Recently, a study based on Reliable Quantitative Structure-Activity Relationship (QSAR) models has investigated the antimicrobial activity of 35 dietary polyphenols against S. aureus, B. subtilis, L. monocytogenes, E. coli, P. aeruginosa and Salmonella enteritidis, and it was proposed a model for the prediction of their activity (Bouarab-Chibane et al., 2019). The antimicrobial activity of polyphenols is not fully elucidated and requires further investigations. Nevertheless, studies have shown that different mechanisms of action potentially may be involved. The antibacterial activity of polyphenols could be mediated through their ability to form complexes with proteins involving nonspecific interactions such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation (Makarewicz et al., 2021). For example, kaempferol-3-rutinoside was demonstrated to inhibit Streptococcus mutans by affecting sortase A, a membrane enzyme that actively plays a crucial role in bacteria adhesion and the invasion of host cells (Kang et al., 2006). Furthermore, polyphenols may interact with both, bacterial cell wall and cell membrane and alternate their function (Makarewicz et al., 2021). For example, a study has shown that epigallocatechin gallate disrupts E. coli cell membrane (Nie et al., 2018). Moreover, polyphenols can influence gene expression regulation. They were found to disrupt DNA, by inhibiting both energy metabolism and DNA synthesis (Makarewicz et al., 2021). In addition, they may act through inhibiting virulence factors. Incubation for 20 h of Cranberry pomace and the flavonol-rich fraction showed bactericidal effects against Salmonella strains, by reducing the expression of virulence genes and cell wall/membrane biogenesis (Makarewicz et al., 2021). Moreover polyphenols have shown inhibitory effects to biofilm formation through interfering with bacterial quorum sensing (Matilla-Cuenca et al., 2020). This antimicrobial effect of polyphenols suggests that a diet enriched with polyphenols compounds could help to maintain or even restore gut homeostasis. Most of above-mentioned bacteria inhabited by polyphenols, are linked with the production of inflammatory cytokines. For example Salmonella typhimurium and C. difficile, causes their production (Lombardi et al., 2018). Moreover, as discussed previously, their abundance has been observed in brain diseases; for example, Pseudomonas aeruginosa with MS (Chen et al., 2016), Escherichia with anxiety (Jiang et al., 2018), Prevotella and Klebsiella species with MD (Lin et al., 2017), Corynebacterium spp with PD (Wallen et al., 2021).I.C.2.3. Polyphenols and modulation of host metabolismIn addition to the effects on gut composition, polyphenols and their gut-derived metabolites can shape the gut microbiota, indirectly, by affecting host metabolism. By contributing to the production of beneficial metabolites, they greatly affect the host health. In vitro studies have demonstrated the impact of polyphenols including 5-CQA, rutin, caffeic acid, quercetin or olive oil on the production of (SCFAs)[START_REF] Mosele | Metabolic and Microbial Modulation of the Large Intestine Ecosystem by Non-Absorbed Diet Phenolic Compounds: A Review[END_REF]. SCFAs production leads to a further increase of beneficial bacteria, and therefore contributes to intestinal homeostasis. The role of SCFAs, in host health was discussed above and the role of polyphenols in their production will be described in the supplementation study. I.C.2.4. Polyphenols and modulation of gut composition in brain diseases Several studies have elegantly addressed the favourable biological effects of polyphenols in several metabolic diseases through the modulation of the composition of gut bacteria (Cheng et al., 2019; Li et al., 2019; Ushiroda et al., 2019; Wang et al., 2019; Yan et al., 2020; Yang et al., 2019). A study with high-fat induced diet provides the evidence on how anthocyanidin extracts by modulating gut microbiota may improve neurologic complications of obesity patients (Marques et al., 2018). In a rat model study, Song and colleagues have shown that 5-CQA could significantly ameliorate depressive-like behaviours by modulating the abundance of specific bacteria. This study showed that treatment with 5-CQA decreased the abundance of Desulfovibrionales, Desulfovibrio, Klebsiella, Burkholderiales and increased Bifidobacterium in gut microflora (Song et al., 2019).However, studies in the field of brain are scarce and further research should be encouraged to give more information on how reshaping gut microbiota through dietary polyphenols may provide positive effects in mental health and brain-related disorders.II.A. Electrochemical studies on 5-CQA and CAOur first objective was to identify the oxidized compounds arising from electrochemical and enzymatic oxidations of 5-CQA and its major gut-derived metabolites CA. Moreover, the generated MS/MS profile will help us to look for possible oxidized compounds occurring by polyphenols-gut microbiota interactions. II.A.1. Study of the electron transfer properties of phenolic compounds Remarkably, polyphenols have drawn attention for their antioxidant properties as described above. Such compounds have shown activity towards inhibition of reactive oxygen/nitrogen species, and modulation of redox signalling pathways (Bucciantini et al., 2021; Crozier et al., 2009; Hussain et al., 2016). The antioxidant process is a complex system that involves electron, proton and radical transfers, among other modes of actions. The reactivity mechanisms brought into play by polyphenols are difficult to observe at the cell level. Thus, many methods for determining in vitro antioxidant capacity have been developed (Apak, 2019). Most of the tests developed are not close to physiological conditions. Nevertheless, even if the values obtained depend on the analysis method and conditions (solvent used, radical studied...) and cannot be extrapolated to the living environment, these in vitro tests have the advantage of highlighting the capacity of a molecule to participate in a radical and/or electron transfer mechanism. Therefore, various in vitro methods have been applied to evaluate the antioxidant activities of these compounds (Gulcin, 2020; Magalhães et al., 2008; Prior et al., 2005). Some methods are based on radical hydrogen atom transfer mechanisms (HAT methods), while others on single electron transfer mechanisms (SET methods). Both types of reactivity can lead to identical reaction products but involve different reaction intermediates. The HAT mechanism of antioxidant relay on ability to transfer hydrogen atom (H) to a peroxyl radical (Eq 1). It should be noted that a HAT process refers to simultaneous proton and electron transfer in the same atomic orbital. It also exists a "sub-HAT" mechanism, the Proton Concerted Electron Transfer (PCET) mechanism in which the simultaneous proton and electron transfer takes place in two different atomic orbitals (Apak et al., 2016; Meyer et al., 2007). Mainly, the HAT-based tests are the Oxygen Radical Absorption Capacity (ORAC), Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP) and Total Oxyradical Scavenging Capacity (TOSC) assays (Munteanu and Apetrei, 2021).

ArOH

  (Muñoz-Rugeles and Alvarez-Idaboy, 2015). This process is particularly interesting in physiological conditions. Indeed, at the physiological pH, due to their pKa range, the phenolic compounds lead to a non-negligible portion of phenolate forms which promote the transfer of electrons[START_REF] Dangles | The Physical Chemistry of Polyphenols: Insights into the Activity of Polyphenols in Humans at the Molecular Level[END_REF][START_REF] Di Meo | Free radical scavenging by natural polyphenols: atom versus electron transfer[END_REF].It should be noted that the predominant mechanism for a given system depends on the structure, the properties of the antioxidant, its solubility in the solvent used and the study parameters (pH, temperature, concentration, and ratio antioxidant/ oxidant). Several studies have shown a difference in the antioxidant capacity of the compounds studied depending on the radical used (Tabart et al., 2009). Therefore, measured antioxidant capacity should always refer to the study context, as total antioxidant capacity may differ from antioxidant properties in biological systems. However, the diversity of antioxidant tests and radicals used, combined with a lack of standardization of these tests, makes it difficult to compare the results obtained between the different studies and the different compounds. Electrochemistry commonly is a useful method which provide interesting elements, in terms of redox potential and also on the mechanisms by which the phenolic compounds could be oxidized. Hence, the oxidation potential values also reflect the antioxidant capacity of the phenolic compounds and have been correlated in some cases with values obtained by other methods. Moreover, they have been described as more accurate than values obtained by the DPPH test (Arteaga et al., 2012; Gomes et al., 2008).

  Nernst equation for a reversible redox couple is: E° (in volts) is the standard potential of the redox system considered, established under standard conditions. with an activity or fugacity (for gases) equal to 1 (aox = ared = 1) at 25°C, following the recommendations IUPAC (International Union of Pure and Applied Chemistry).When we are not in the standard conditions, the formal potential or apparent standard potential E'° is taken into account. This E'° is measured experimentally in solutions containing equal concentrations of the species Ox and Red species. E'° considers the ionic strength, pH, possible complexation and precipitation phenomena involving all the species in the Nernst equation.In solution, the reducing capacity of a molecule may be evaluated by its redox potential (Eredox). Indeed, the lower the value of the potential, the lower the energy required to remove R perfect gas constant (8.314 J. K -1 . mol -1 ) F Faraday constant (96 485 Coulombs) T (K) absolute temperature n number of electrons involved Expression at 25 °C (298 °K) ln = 2.303 x log10 an electron, thus the higher the reducing capacity of the compound. In fact, the redox potential of the couple phenol/phenoxyl radical being relatively low, reactions of hydrogen atom transfers and/or rapid electrons with ROS can take place and constitute one of the main antioxidant mechanisms of phenolic compounds (Cheng et al., 2002). The techniques for measuring these potentials are mainly based on the measurement of electron transfers such as pulsed radiolysis or by electrochemical techniques such as cyclic voltammetry (CV) or square wave differential voltammetry (Square Wave Voltammetry, SWV) (Sochor et al., 2013; Ziyatdinova et al., 2013). In all cases, the measurement of the potential depends on the medium in which the compounds are studied, on the pH and on the temperature. II.A.1.3. Electrochemistry Electrochemical techniques such as voltammetry, chronoamperometry and spectroelectrochemistry UV visible, are considered powerful analytical tools to study electron transfer reactions (Sochor et al., 2013). They can help to understand the reactivity in vivo of molecules with redox activity by studying the processes triggered by electron transfer,accessing certain thermodynamic (redox potential) and kinetic parameters and by creating physiological radicals (e.g., O2 •-) directly at the electrode (unlike the tests mentioned above such as DPPH), thus, allowing the analysis of the radicophilic properties of the substrate.Polyphenols being electroactive species, the study of their oxidation mechanism by the use of electrochemical tools is rapid, sensitive, easy to implement, reproducible, and does not use radicals, the electrode serving as a oxidant[START_REF] Cheng | Phenolic antioxidants: electrochemical behavior and the mechanistic elements underlying their anodic oxidation reaction[END_REF].PrinciplesAn electrochemical reaction is defined as a heterogeneous electron transfer occurring between an electrode and a species in solution. The electrochemical reaction occurs at the electrode when it is brought to a potential different from its equilibrium potential (overpotential η). This overpotential can be related to the apparent normal potential by Nernst's law (see equation below). The redox system will then evolve towards a new equilibrium state by changing the concentrations of oxidant (C sol ox) and reductant (C sol red). The electron transfer reactions can be studied by three-electrode techniques (working or indicator electrode, reference electrode, auxiliary electrode) connected to a potentiostatgalvanostat which allows control of the potential (in the working-reference electrode circuit) and current measurement (in the working-auxiliary electrode circuit) (Sochor et al., 2013). The direction of the current generated depends on the value of the potential imposed on the working electrode in relation to the reference electrode. The production of an electrochemical reaction follows a quantitative law (Faraday's Law): N number of moles of oxidant or reductant electrolysed Q the total electrical charge passed during electrolysis F = 96485 C mol -1 , the Faraday constant n number of moles of electron exchanged Voltammetry In these techniques, voltammograms representing I = f (E) are generated by a potential sweep by gradually modifying the setpoint voltage controlled by the potentiostat and the measurement of the corresponding electrolysis current. In this system, electrolysis occurs in the low-volume diffusion layer (A.δ(t)). The concentration of the species studied is therefore not modified in solution (Figure 20).

Figure 20 .

 20 Figure 20. Representation of diffusion layer (A: area of the electrode; δ (t): diffusion layer)

Figure 22 .

 22 Figure 22. Spectroelectrochemistry UV-VIS setup

II.A. 2 . 1 .

 21 Cyclic voltammetryThe electrochemical behaviour of 5-CQA and CA was investigated by cyclic voltammetry in 0.1 M PBS at pH 5.8. The cyclic voltammogram of both phenolic compounds is shown in Figure23, where one oxidation and one reduction peaks are observed. 5-CQA and CA possess in their structure a catechol moiety with two -OH groups in the ortho-position, which is known to oxidize into ortho-quinone form by a transfer of two-electron-two-proton mechanism (Namazian and Zare, 2005; Šeruga and Tomac, 2014; Yardım, 2012). As shown in Figure23, 5-CQA exhibited an anodic peak at Epa = + 0.55V/Ag/AgCl and CA one at Epa = + 0.38V/Ag/AgCl. Then, on the reverse scan, the reversible reduction of the quinone its catechol form is observed (Epc = + 0.11V/Ag/AgCl Epc = + 0.14V/Ag/AgCl, respectively

Figure 23 .

 23 Figure 23. Cyclic voltammograms of 5-CQA (a) and CA (b) 2 mM in pH 5.8 at 0.1 M in phosphate buffer vs. Ag/AgCl reference electrode using a GCE working electrode, scan rate 0.1 V.s -1 , room temperature.

Figure 24 .

 24 Figure 24. In situ thin layer UV vis spectra of a) 5-CQA and b) CA in function of the electrolysis time.

  .

Figure 25 .

 25 Figure 25. Base peak chromatograms of: a) electrolyzed solution of 5-CQA (2 mM); b) oxidized solution of 5-CQA by PPO

  electrochemical oxidation. Moreover, less ions at m/z 705 were observed enzymatically vs electrochemically with the presence of 9 peaks(5, 6, 10, 14, 15, 18, 19, 21, 23) and the absence of the peaks 11-13, 16-17, 20-22 and 24. In addition, two peaks 8 and 9 with m/z 723 were characteristic of enzymatic oxidation of 5-CQA, compared to others 3 and 4 which were also observed in the electrochemically oxidized sample. The oxidized compounds formed both electrochemically and enzymatically have shown the same UV and MS/MS patterns (Scheme 2). UV spectrum of ions exhibited a maximum absorbance in 280-320 nm range for all oxidized compounds. Most of the ions at m/z 705 have shown the MS/MS fragment with m/z 513 corresponding to the loss of one quinic acid moiety. Only peak 10, formed enzymatically, showed a different MS/MS spectrum, with the most intense fragment ion at m/z 321, corresponding to the loss of both quinic acid moieties. The ion with m/z 703 showed a MS/MS fragmentation with m/z 511. Previous studies carried out by Guyot et al. led to the identification of oxidized compounds after enzymatic oxidation of 5-CQA by PPO. About ten products at m/z 705 (706 Da) corresponding to dehydrodimers of 5-CQA were observed. While the compounds with m/z 703 (704 Da) were identified as reoxidized forms of 5-CQA dehydrodimers, ions at m/z 723 were assigned to hydrated 5-CQA dehydrodimers (724 Da) (Bernillon et al., 2004; Wong-Paz et al., 2015). Moreover, the Guyot's team has recently purified using centrifugal chromatography partition (CPC), ten 5-CQA dehydrodimers (706 Da) (Castillo-Fraire et al., 2019), and has determined the structure by 1 H and 13 C one-and two-dimensional NMR spectroscopies of seven 5-CQA dehydrodimers with dihydrobenzofuran (type I), benzodioxane (type II) or dihydronaphthalene (type II) skeleton for six of them and with a symmetric dicatechol structure (type IV) for the last one Scheme 2 (Castillo-Fraire et al., 2022).

  Scheme 2. Mechanisms of formation and structures of oxidized products derived from 5-CQA (adapted from(Castillo-Fraire et al., 2019) and (Castillo-Fraire et al., 2022)

1- 4

 4 type ring nucleophilic condensation (type a, like C-C dimerization) or by condensation between semi-quinone radicals (types b and caffeicin-like dimers c, d) (see Scheme 3) (Pati et al., 2006; Weber et al., 2019), were not detected, neither electrochemically nor enzymatically. However, some peaks (17-23) obtained electrochemically and those (18, 21, 22) afforded enzymatically exhibited m/z 313, suggesting that they could be generated from CA dimers (Pati et al., 2006) and correspond to [M-CO2-H] -ions. Peaks 17, 18, 20 demonstrated a same MS/MS pattern at m/z 269 arising from further decarboxylation and indicating their belong to C-C dimers of CA (type a). It is interesting to note that it is the dimer 18 which was the most abundant product in each oxidized sample. Peak 19 may also correspond to a C-C dimer as reported by Pati et al. Moreover, the same MS/MS spectra (269, 179, 177, 135 and 133) were detected for peaks 21-23. These molecular fragmentations come either by CO2 loss, directly or combined with a retro Diels Alder and could lead to the determination of their structures as caffeicin-like dimers of CA (c) (Pati et al., 2006). Some peaks (12-16) detected in the enzymatic study corresponded to high molecular weight metabolites (e.g m/z 383, 397, 427, 441, and 471) which are less polar than CA.

Figure 26 .

 26 Figure 26. Base peak chromatograms of: a) electrolyzed solution of CA (2 mM); b) oxidized solution of CA by PPO

Figure 27 .

 27 Figure27. A) The well-known mechanism of antioxidants by modulating redox state and redox signalling pathways: B) potentially bioactivity of oxidized metabolites obtained via free radical scavenging[START_REF] Hunyadi | The mechanism(s) of action of antioxidants: From scavenging reactive oxygen/nitrogen species to redox signaling and the generation of bioactive secondary metabolites[END_REF]

Figure 28 .

 28 Figure 28. Scanning electron microscopy of Lactobacillus reuteri strain[START_REF] Patman | Gut microbiota: The difference diet makes to metabolites and microbiota[END_REF]

Figure 29 .

 29 Figure 29. Scanning electron microscopy of Bacteroides strain

Figure 30 .

 30 Figure 30. Scanning electron microscopy of Bifidobacterium strains (Rouvet, 2004).

2 .

 2 The roles in intestinal health of L. reuteri, B. fragilis and B. longum The well-studied probiotic bacterium, L. reuteri is found in different body sites, including the gastrointestinal tract, urinary tract, skin and breast milk (Mu et al., 2018). The abundance of L. reuteri varies among different individuals. Several beneficial effects of L. reuteri have been reported. Due to its antimicrobial activity, L. reuteri can inhibit the colonization of pathogenic microbes and remodel the commensal microbiota composition in the host. Moreover, L. reuteri has shown immunomodulatory effects. For example, some strains can reduce the production of pro-inflammatory cytokines while promoting regulatory T cell development and function. Finally, considering its ability to enhance the intestinal barrier, the colonization of L. reuteri may decrease the microbial translocation from the gut lumen to the tissues (Mu et al., 2018).In the micro-flora of the human gastrointestinal tract, B. fragilis, is an anaerobic commensal constituting only 1-2% of it, and it represents the predominant anaerobic isolate in cases of intra-abdominal sepsis and bacteraemia as mentioned above. B. fragilis can play two roles in the host (Yekani et al., 2020). As a friendly commensal, this strain must be able to establish itself in the host intestinal mucosa, to utilize and process polysaccharides for use by the host, and to resist to the noxious effects of bile salts. As pathogen, it must be able to attach itself to the site of infection, evade killing mechanisms implemented by host defense or with an antimicrobial treatment and produce factors that damage host tissue. The cell envelope of B. fragilis, likewise, must be able to be aggressor, defender and strategist in allowing the organism to establish itself in the host-whether as friend or foe[START_REF] Pumbwe | The Bacteroides fragilis cell envelope: Quarterback, linebacker, coach-or all three?[END_REF].Bifidobacteria are gut commensals and are among the first microbes to colonize the human gastrointestinal tract. Among the probiotic features and functions that have been attributed to Bifidobacteria are the production and/or release of vitamins B, antioxidants and polyphenols; maturation of the immune system during early life and preservation of immune homeostasis during life; preservation of gut barrier functions and protection against pathogens by production of bacteriocins, decreasing luminal pH by acids production, and blocking the adhesion of pathogens to the intestinal mucosa(Gagnon et al., 2015). Another important function of the bifidobacterial genus is their contribution to gut homeostasis and host health in the production of acetic and lactic acids during carbohydrate fermentation, organic acids that in turn can be converted into butyric acid by other colon bacteria through cross-feeding interactions. In this context, B. longum species has a long history of safe use as probiotic adjunct in foods and in pharmaceutical (O'Callaghan and van Sinderen, 2016).

  molecules have shown the ability to lower oxidative stress involved in many diseases and positively modulate redox signalling pathways (Zhang and Tsao, 2016). Luca et al. has recently described the notion of "low bioavailability/high bioactivity paradox of polyphenols" highlighting the interest of their intestinal metabolites on human health (Luca et al., 2020). Considering their superiority in terms of presence in systemic circulation compared to parent polyphenols, further insights focus nowadays on the pivotal role of microbiota to generate bioavailable low molecular weight metabolites (Pasinetti et al., 2018a).These gut-derived metabolites have exhibited biological activities and have shown ability to reach the brain (Carregosa et al., 2019b; Johnson et al., 2019; Yamasaki et al., 2020). Indeed, studies have put them in the spotlight as promising approach to prevent and attenuate many pathologies such as neurodegenerative diseases (Carregosa et al., 2019b; Yamasaki et al., 2020). Among dietary polyphenols, the hydroxycinnamate derivative 5-O-caffeoylquinic acid (5-CQA) according to IUPAC name, known as chlorogenic acid, is one of the main phenolic compounds in daily diet, being present in various fruits and vegetables (Clifford, 1999; Lu et al., 2020). This highly functional compound has been shown to have high benefits for human health (i.e. cardioprotective, gastrointestinal protective, hepatoprotective, neuroprotective properties etc..) (Lu et al., 2020). Pharmacokinetic profiles of 5-CQA have been already described in humans (Clifford et al., 2017). After intake, one third is absorbed in small intestine and majority reaches the colon in intact form. At this stage, intestinal microbiota with cinnamoyl esterase activity biotransform 5-CQA into caffeic acid (CA) and quinic acid, which further undergo their subsequent transformation into various microbial metabolites including m-coumaric acid and hydroxylated derivatives of phenyl propanoic, benzoic, and hippuric acids (Clifford et al., 2017). These metabolites are bioavailable in the systemic circulation and achieve the targeting organs where they can exert their biological effects (Clifford et al., 2017; Lu et al., 2020). Indeed, the biotransformation of 5-CQA is dependent on the ability of bacterial species to produce this esterase as the first step. Hence, some bacteria species were already identified as producer of this specific enzyme such as Escherichia coli, Bifidobacterium lactis, Lactobacillus gasseri (Couteau et al., 2001b) or L. johnsonii (Bel-Rhlid et al., 2013). In this work, we have investigated the biotransformation of 5-O-caffeoylquinic acid by three gut-bacteria species cultivated alone: Lactobacillus reuteri (Firmicutes), Bacteroides fragilis (Bacteroidetes) and Bifidobacterium longum (Actinobacterium), belonging to the dominant bacterial phyla of the gut microbiota. The biotransformed extracts were analysed by HPLC-DAD-ESIMS 2 and the data were subjected to MZmine 2.53 and a further treatment creating a molecular network (MN). It is interesting to note that gut microbiota polyphenol biotransformation encompassing hydrolysis, reduction and de-or di-hydroxylation reactions are well known (Espín et al., 2017; Vollmer et al., 2017) but there is a lack of information on possible oxidative pathways other than 𝛼or 𝛽-oxidations. For example, one study led to the characterization of oxidation of one metabolite of 5-CQA, 3-(4'-hydroxyphenyl) propanoic acid, into 4-hydroxybenzoic acid, using an in vitro experimental gastrointestinal dialysis model (Breynaert et al., 2015). Hence, electrochemical and enzymatic methods were adopted to generate oxidized compounds of 5-CQA and CA in order to characterize and compare their LC-MS profiles to those of the biotransformed extracts. This strategy allowed to highlight an intriguing oxidative pathway of cinnamic acids microbiota conversion. II.B.2. 2. Results and discussion a) Biotransformation studies of 5-CQA The resting cell method (Julsing et al., 2012) was applied for the biotransformation of 5-CQA using three gut bacteria L. reuteri, B. fragilis and B. longum. Based on the growth curve of these bacteria monitored by the measure of OD600nm, the biotransformation reaction was performed during 24 h when the stationary phase was reached. PBS was chosen as a solvent for 5-CQA and CA since it was previously described as closer to the intracytoplasmic bacterial composition (Mortelé et al., 2019). The biotransformation step was done at pH 5.8, close to the pH found in colon. 5-CQA was dissolved in PBS at 1 mM, a concentration close to those in the colon after coffee or fruits intake (Clifford, 1999). After centrifugation, the pellet and the supernatant were separately treated using ethyl acetate to obtain two extracts which were further analysed by LC-UV-ESI-MS 2 in negative mode. The base peak chromatograms of the control (5-CQA incubated at 37°C) and of the SN extracts after 5-CQA biotransformation by the three bacteria are reported in Figure 31. While L. reuteri was able to biotransform 5-CQA, none biotransformed metabolites were detected after bioconversion by B. fragilis and B. longum Figure 31. To validate these results, we searched in silico for enzyme responsible of CQA biotransformation named cinnamoyl esterase or feruloyl esterase (EC.3.1.1.73), which was already found in L. johnsonii (Bel-Rhlid et al., 2013) (accession number NC_005362). A genomic comparison between the gene already described in L. jonhsonii and those present in our strain L. reuteri DSM 20016 (GCA_001436151 showed 70.7% of similarity with the geneLocus Tag named LREU_RS08790 (Figure 32). Nonetheless, this gene was not found in B. fragilis and B. longum, suggesting that the results from biotransformation confirmed by LC-ES-MS 2 are in line with search in silico. Research on Pfam highlighted the presence of the same domain, hydrolase_4, serine aminopeptidase, S33 in these two genes which belong to the Alpha/Beta hydrolase fold family (El-Gebali et al., 2019). This catalytic domain is found in a very wide range of enzymes like esterase. This cinnamoyl esterase led the hydrolysis of 5-CQA into CA and quinic acid. CA was well detected in LC-ESI-MS 2 chromatogram and quinic acid, being too polar, was either not extracted by ethyl acetate or not retained on the column (Figure 31) The peak 3 was identified as cis-5-O-caffeoylquinic acid as suggested by Clifford et al (Clifford et al., 2008) and confirmed after UV irradiation in a UV-quartz cell during 1 h of 5-CQA standard (Figure 33).

Figure 31 .

 31 Figure 31. Base peak chromatograms in negative mode of a) 5-CQA incubated at 37°C in PBS at pH 5.8, b) SN extracts after 5-CQA biotransformation by L. reuteri in PBS, c) SN extracts after 5-CQA biotransformation by B. fragilis in PBS, d) SN extracts after 5-CQA biotransformation by B. longum in PBS. (1) 5-CQA, (2) isomer of 5-CQA (3) cis-5-O-caffeoylquinic acid, (4) esculetin, (5) trans-CA, (6) cis-CA. *compounds with retention time (RT) confirmed after injection of analytical standards

  found either simultaneously in 5-CQA sample and in one biotransformed extract or in SN L. reuteri biotransformed extract alone) as quinic acid after dereplication but with a very low score (cos = 0.4) (data not shown). CA and coumaroyl quinic acid have been already reported after 5-CQA in vitro biotransformation by gut microbiome (Mortelé et al., 2019; Tomas-Barberan et al., 2014).

Figure 34 .

 34 Figure 34. Molecular network analysis in negative mode of extracts and samples. 1 and 2: pellet and SN extracts from L. reuteri incubated in medium; 3 and 4: pellet and SN 5-CQA biotransformed extracts from L. reuteri; 5 and 6: pellet and SN extracts from L. reuteri incubated in PBS; 7: 5-CQA incubated at 37°C with PBS; 8: PBS control. Only nodes specific from biotransformed extracts (green color) with cos > 0.9 and related to 5-CQA structure were annotated

  of trans-CA after oxidation through an isolated rat liver[START_REF] Gumbinger | Metabolism of Caffeic Acid in the Isolated Perfused Rat Liver[END_REF]. Our result underlines for the first time this specific mechanism by L. reuteri DSM 20016 strain leading to the formation of a coumarin, esculetin, after ring closure of cis-caffeoyl quinone, via an interesting oxidative pathway of 5-CQA. Table 5. LC-DAD-ESI-MS 2 metabolites identified or detected in SN extract after 5-CQA biotransformation by L. reuteri. A putative 5-CQA biotransformation pathway by L. reuteri leading to esculetin is shown in Scheme 4. Due to the presence in the standard of all the two geometric 5-CQA isomers (ID 3 and 9), the first step could be, after ester cleavage, a geometric isomerization of trans-CA into its cis isomer which was already reported by photoisomerization (Parrino et al., 2016). This evidence could be supported by the detection of cis-CA (ID 8) and suppose the presence of a cis-trans isomerase in L. reuteri enzymatic machinery. This kind of enzyme has been already reported in Lactobacillus (Kishino et al., 2013; Sun et al., 2020) and after genomic comparison an ortholog sequence in L. reuteri studied strain (DSM 20016, GCA_001436151) was found (Figure 35). The other possibility of the presence of cis-CA could come from hydrolysis of the residual cis-5-O-caffeoylquinic acid (ID 9) by cinnamoyl esterase. Similarly with the process of oxidative cyclization of cis-CA to esculetin after photocatalytic isomerization proposed previously (Parrino et al., 2016), the next step corresponds to the formation of caffeoyl quinone probably by an oxidase as the biotransformation was done under anaerobic conditions. Previous studies have reported the enzymatic conversion of cis CA into esculetin catalysed by an oxidase, previously named phenolase ([START_REF] Sato | Varietal Differences in the Phenolic Content and Superoxide Radical Scavenging Potential of Wines from Different Sources[END_REF][START_REF] Satô | Metabolism of phenolic substances by the chloroplasts-III.: Phenolase as an enzyme concerning the formation of esculetin[END_REF]

Figure 37 .

 37 Figure 37. PDA chromatograms at 320 nm of a) 5-CQA incubated with PBS, b) 5-CQA incubated with BHI, c) SN extracts after 5-CQA biotransformation by L. reuteri in PBS, d) pellet extracts after 5-CQA biotransformation by L. reuteri in BHI, e) CA incubated with PBS, f) CA incubated with BHI, g) pellet extracts after CA biotransformation by L. reuteri in BHI. (1) 5-CQA, (2) isomer of 5-CQA (3) cis-5-O-caffeoylquinic acid, (4) esculetin, (5) trans-CA, (6) cis-CA.

(

  Cheynier and Moutounet, 1992a;[START_REF] Pati | Characterization of caffeic acid enzymatic oxidation by-products by liquid chromatography coupled to electrospray ionization tandem mass spectrometry[END_REF].II.B.2.3. ConclusionsIn summary, 5-CQA was differently biotransformed following the gut bacteria strains used. Molecular network analysis led to the identification, after 5-CQA biotransformation by L. reuteri, of one oxidized derivative of cis-CA, caffeoyl quinone, which was stabilized into esculetin its ring closure form. The inability of the two other studied gut bacteria to biotransform 5-CQA supports one more time the importance of interindividual differences of microbiota composition (Frolinger et al., 2019). The comparison with other oxidation methods such electrochemical and enzymatic ones confirm the formation of caffeoyl quinone followed by a ring closure into esculetin. The electrochemical and enzymatic studies also led to show that no other oxidation products of 5-CQA and CA were formed during biotransformation.These findings put also on the spotlight the interesting and specific enzymatic machinery of L. reuteri encompassing a cinnamoyl esterase, isomerase and oxidase leading to a coumarin compound. Further studies should be undertaken with the aim to identify the potential biological effects of esculetin on gut-microbiota axis and will give more insights whether this metabolite is more active than the parent one, an evidence that we have already demonstrated with olive oil phenol compounds (Lambert de Malezieu et al., 2019). Chlorogenic acids formulated as synbiotics (Swanson et al., 2020) with L. reuteri may be finally promising candidates in human health application. II.B.3. Biotransformation of 5-CQA by co-culturing in vitro two gut bacteria, L. reuteri and B. fragilis

  reuteri and B. fragilis, were co-cultured. Such studies will provide information on the potential influence of bacterial interactions one the community during biotransformation. II.B.3.1. Context of the study According to the literature , the implications of the gut microbiota in health and disease nowadays is unquestionable (Cani, 2018; Codagnone et al., 2019; de Vos et al., 2022; Ding et al., 2019; Fan and Pedersen, 2021; Schächtle and Rosshart, 2021). It is well established that gut microbiota as a dynamic ecosystem communicates constantly with the host, providing the gut homeostasis (Bäckhed et al., 2005; Maynard et al., 2012; Perry and Arsenault, 2022). Indeed, the interaction network in the gut involves the triangle of : commensalpathogen , commensal-host; and pathogen-host interactions (Khan et al., 2021). Most studies have focused on gut microbes and their host interactions and their outcomes related to health and diseases (Ayres, 2016; Yongyan Chen et al., 2021; Horne et al., 2019; Parker et al., 2018; Tsolis and Bäumler, 2020). However, little is known about the complex interplay between bacteria in the gut microbial community (Khan et al., 2021). Notably, interaction patterns mainly include competition for nutrients and space, followed by exchange of nutrients, enzymes, and genetic materials (Kern et al., 2021) and/or the capacity of co-aggregation and involvement in biofilm formation (Palencia et al., 2022

  expression of virulence factors (Bäumler and Sperandio, 2016; Khan et al., 2021; Vogt et al., 2015). For example, Gharbi et al. (2018) have shown that Lactobacillus fermentum and Lactobacillus plantarum, isolated from the human microbiota, were able to reduce the adhesion to HT29 cells of the pathogens such as Escherichia coli, Cronobacter sakazaki and Salmonella enterica (Gharbi et al., 2018). Besides that, recently, specific interactions such as cross-feeding and quorum sensing have received increasing attentionin bacterial interaction studies (Jo et al., 2021). Quorum sensing is a chemical communication between bacteria, in which individual bacteria in a community, coordinate their behavior in response to environmental changes of density and composition (Mukherjee and Bassler, 2019). Thus, depending on the circumstances, bacteria modify metabolic pathways, the pattern of virulence factors and/or the capacity of biofilm formation by self-regulating the expression of relevant genes (Prescott and Decho, 2020).

Figure 38 .

 38 Figure38. A schematic illustration of cross-feedings, including metabolite, substrate, mutualism, and augmented forms[START_REF] Smith | Microbes and the Mind: How Bacteria Shape Affect, Neurological Processes, Cognition, Social Relationships, Development, and Pathology[END_REF]

  Figure 39 .

Figure 39 .

 39 Figure 39. (A): Number of bacteria (CFU.mL -1 ) versus time: (A): culture evulution of L. reuteri in mono and co-culture; (B) B. fragilis in mono and co-culture; (C)

Figure 40 .

 40 Figure 40. Base peak chromatograms in negative mode of a) 5-CQA biotransformation by L. reuteri: b) 5-CQA biotransformation by a co-culture of L. reuteri and B. fragilis. (1) 5-CQA, (2) esculetin (3) trans-CA, (4) cis-CA, (5) cis-5-CQA. *Compounds with retention time (RT) confirmed after injection of analytical standards

Figure 41 .

 41 Figure 41. Calibration curves for 5-CQA, CA and esculetin in MeOH

  biotransformation of dietary components (Adak and Khan, 2019). In addition, it is considered as a key node in the communication between gut and brain. Moreover, numerous studies have documented significant changes of gut microbiota composition and function associated to brain diseases and other conditions such as obesity, diabetes, non-alcoholic liver diseases, cardiometabolic diseases[START_REF] Cani | Human gut microbiome: hopes, threats and promises[END_REF][START_REF] Cryan | The gut microbiome in neurological disorders[END_REF] as already discussed before. Given all of this, it is important to investigate how to regulate the microbiota composition and function as a potential approach to alleviate symptoms, and positively influence the treatment of neurodegenerative diseases[START_REF] Di Meo | New Therapeutic Drugs from Bioactive Natural Molecules: The Role of Gut Microbiota Metabolism in Neurodegenerative Diseases[END_REF] Filosa et al., 2018b). Indeed, the dietary patterns are one of the main factors shaping the gut microbiota (Lombardi et al., 2018; Zmora et al., 2019). Moreover, the link between diet, gut microbiota and health, including good mental health and positive impacts on brain diseases has been demonstrated (Berding et al., 2021). Furthermore, there is increasing awareness of the implications of eating habits on well-being (Watson, 2020b). Therefore, through diet and nutrition, scientific investigations aim to improve mood and mental health, enhance cognitive ability and have a positive effect on brain diseases (Adan et al., 2019). In the context of diet, accumulating data suggest that polyphenols represent a more promising nutraceutical strategy than can beneficially reshape gut microbiota (Bolte et al., 2021; Gutiérrez-Díaz et al., 2020; Plamada and Vodnar, 2021), which is not surprising givenhighly concentrated dietary bioactive components possessing antioxidant properties (Silva and Pogačnik, 2020; Tresserra-Rimbau et al., 2018). As already mentioned, humans consume up to 1 g polyphenols per day, and as an illustration compared to other antioxidant molecules, this corresponds to ten times more than the amount of vitamin C and /or one hundred times more than those of vitamin E and carotenoids (Scalbert et al., 2005a). Once polyphenols are consumed through the diet, most of them reach the colon in their native form, as discussed above. Reciprocal interactions occurring in the gut also involve the impact of polyphenols on gut microbiota. Thus, the unabsorbed part, as well as polyphenols gut-derived metabolites, greatly affect the gut microbiota composition and function (Ozdal et al., 2016). The modulation of gut microbiota by polyphenols has been established so far. As discussed previously, the beneficial impact relies on the prebiotic effect capacity or inhibition of pathogens (Plamada and Vodnar, 2021). Moreover, polyphenols can modulate the metabolism of gut microbiota by influencing the production of SCFAs and BCFAs (Mosele et al., 2015). Acetic, propanoic, butyric, formic and valeric acids are mainly produced from dietary fermentation by the gut microbiota (Koh et al., 2016). Specifically, polyphenols induce production of SCFAs by promoting the growth of such known SCFAs producing bacteria of (Parkar et al., 2013; Unno and Osakabe, 2018). Moreover, animal polyphenol supplementation studies have shed more light on the hypothesis that by regulating gut microbiota, polyphenols could positively impact health, mainly in the context of metabolic diseases. These studies revealed an increase of SCFAs in addition to the changes in gut microbiota composition (Anhê et al., 2018; Li et al., 2019; J. Liu et al., 2019; Van Hul et al., 2018). In addition, these metabolites have been reported to exert several positive effects in the human health, due to their key role in gut-brain axis crosstalk. Some reports have highlighted the potential effects of SCFAs on neurological disorders (Arnoldussen et al., 2017; Chen et al., 2022; Gonzalez et al., 2010; Kim et al., 2020; van de Wouw et al., 2018) et al., 2017). Besides that, data on humans still are lacking. However, understanding how SCFAs arederived from polyphenols-gut interactions, could help develop a potential strategy to target the gut-brain axis through the dietary intervention (Dalile et al., 2019a). Contrary, high levels of BCFAs are potentially prejudicial to gut health (Bloomgarden, 2018; Goffredo et al., 2017; Shi et al., 2021) as some correlations have been observed with diabetes (Bloomgarden, 2018), obesity (Goffredo et al., 2017), depression (Szczesniak et al., 2016) and Rett Syndrome (Borghi et al., 2017).

  untreated L. reuteri or untreated B. fragilis. The effect of 5-CQA on L. reuteri and B. fragilis growth was determined by comparing the growth parameters of the cultures treated with different 5-CQA concentrations to those of the untreated ones. These results correspond to the mean of values obtained from the three experiments. The size of the standard deviations shown can beexplained by the cultural requirements of anaerobic bacteria. In fact, these cultures are very stringent in terms of the experimental conditions. a) Effects of 5-CQA on B. fragilis growth Untreated B. fragilis has exhibited a growth culture with a maximum reached at T6h

Figure 43 .

 43 Figure 43. Number of B. fragilis (CFU.mL -1 ) versus time of culture (B): growth kinetics of B. fragilis in BHI standard medium or under different concentrations of 5-CQA. The values reported are means ± SD of three independent experiments

Figure 44 .Figure 47 .

 4447 Figure 44. (A): Number of L. reuteri (CFU.mL -1 ) versus time of culture (B): growth kinetics of L. reuteri in BHI standard medium or under different concentrations of 5-CQA. The values reported are means ± SD of three independent experiments

  sample analyzed and b) the appearance of 1.3-propanediol and 2pyrrolidone production at T6 (depicted in Figure49at T30 as example). In fact, the 2pyrrolidone was far less produced by L. reuteri in comparison with B. fragilis at each time sampling. In conclusion, no representative impact in terms of metabolites production was also noted when L. reuteri cultures were supplemented by 5-CQA.

Figure 48 .Figure 49 1 .

 48491 Figure 48. GC-MS profiles obtained at T0 for L. reuteri: A) in standard condition (BHI = 25 μg.mL -1 of hemin); B) with 5-CQA at 50 µM. (1) hydroxyacetone , (2) acetic, (3) propanoic, (4) isobutyric,(5) butyrolactone, (6) butyric acid

Figure 51 .

 51 Figure 51. Biotransformation pathway of glycerol to 1,3-propanediol by L. reuteri. The enzymes involved in this process includes: [1] B12-dependent glycerol/diol dehydratase , [2] 1,3-propanediol oxidoreductase, [3] propionaldehyde dehydrogenase , [4] phosphotransacylase , and [5] propionate kinase (Dishisha et al., 2014) Abbreviations: 3HPA (3-hydroxypropionaldehyde), 1,3 PDO (1,3propanediol), 3HP (3-hydroxypropionic acid)

(

  Luise et al., 2020).The other metabolite, hydroxyacetone, is produced from methyl glyoxal, a pathway already described for lactic bacteria (Hatti-Kaul et al., 2018). Butyrolactones, such as GBL, are involved in quorum sensing, the cell-to-cell communication processes (Du et al., 2011).

(

  Rodríguez-Daza et al., 2021). As shown in Figure 52 prebiotic effect of polyphenols could be mediated through the activation of polyphenol-associated enzymes (PAZymes). Briefly, a gut bacterium can biotransform a polyphenol (M1) which could be subjected to metabolic utilization, as a source of energy. Then M1 could be converted in a trophic network by other PAZymes bacteria which could lead to the generation of new metabolites. Overall, all this cascade could have positive impact in intestinal barrier integrity, consequently in human health as shown in Figure 52 (Rodríguez-Daza et al., 2021).

Figure 52 .

 52 Figure 52. Overview of proposed duplibiotic nature of dietary polyphenols, including their prebiotic and antimicrobial effects on the gut microbiota (Rodríguez-Daza et al., 2021)

Figure 53 .

 53 Figure 53. PDA at 320 nm of supplementation of 5-CQA (1 mM) in L. reuteri culture (SN extract) at T30: (1) 5-CQA (2) esculetin (3) trans-CA, (4) cis-CA. *compounds with retention time (RT) confirmed after injection of analytical standards

Figure 54 .

 54 Figure 54. Illustrative picture of quadruple cellular model, used in our study

  Figure55). The mean values and SD were calculated from three independent measurements. As shown in Figure55

Figure 55

 55 Figure 55 no differences were observed in the presence of any polyphenols at all concentrations used compared to the untreated cells. These data indicated that of none polyphenols used at these concentrations induced cytotoxicity in the cell model used, suggesting that the conditions studied were appropriate for further investigation.

Figure 55 .

 55 Figure 55. Effects of polyphenols at 2.5 µM, 50 µM and 1 mM on quadricellular cell viability measured by the release of LDH after incubation for 24 h

Figure 56 .

 56 Figure 56. Effects of polyphenols at 2.5 µM, 50 µM and 1 mM on TEER of cells after incubation for 24 h

Figure 57 .

 57 Figure 57. Effects of polyphenols at 2.5 µM, 50 µM and 1 mM on relative expression of occludin after incubation for 24 h. ns: not significantly different

  [START_REF] Bo | Systematic Review on Polyphenol Intake and Health Outcomes: Is there Sufficient Evidence to Define a Health-Promoting Polyphenol-Rich Dietary Pattern?[END_REF][START_REF] Williamson | The role of polyphenols in modern nutrition[END_REF]. These compounds have shown a plethora of bioactivities, mainly mediated by their antioxidant properties and regulation of various redox signalling pathways. The French paradox has drawn the attention of the scientific community toward these molecules. However, their biological activity goes beyond classical exploration as antioxidant molecules(Filosa et al., 2018b;[START_REF] Serra | Dietary polyphenols: A novel strategy to modulate microbiota-gut-brain axis[END_REF]. The different aspects of the study of these molecules will allow for a better understanding of their roles in humans. Electrochemistry is useful tool for studying the antioxidant properties of polyphenols. Moreover, molecules that can arise in redox processes have attracted the attention of scientists, because of their potential positive bioactivities in humans (Lambert de Malezieu et al., 2019; D.Liu et al., 2019). However, there is still a lack of information on the presence of possible oxidized compounds from this type of reaction in the human body. Indeed, there is still no MS/MS database that could help in the identification of such compounds by comparing the MS/MS profiles.With the main objective, of enriching this type of database, we set out to identify the oxidized metabolites obtained after electrochemical and enzymatic oxidation of one of the main dietary polyphenols, 5-CQA and its major gut-derived metabolite CA. The oxidation mechanism of these compounds involves two protons and two electrons is well known (Šeruga and Tomac, 2014).Moreover, some enzymatically generated oxidized compounds from 5-CQA and CA have been already identified (Castillo-Fraire et al., 2019;[START_REF] Pati | Characterization of caffeic acid enzymatic oxidation by-products by liquid chromatography coupled to electrospray ionization tandem mass spectrometry[END_REF]. To the best of our knowledge, there is no data identifying these metabolites using electrochemical studies, which was our first objective. To this end, the UV-Vis spectroelectrochemistry of the two phenolic compounds was explored and the MS/MS profiles of the oxidized solutions were characterized. LC/MS 2 analysis of the electrolyzed solutions of 5-CQA led to the identification of one ion that was identified as a reoxidized form of 5-CQA dehydrodimers with m/z at 703, 17 ions with m/z 705 (MW, 706 Da) corresponding to dehydrodimers of 5-CQA (type I-III) (also named caffeicin-like dimers) and three hydrated dehydrodimers of 5-CQA with m/z 723. These findings are in line with previous data on the enzymatic oxidation of 5-CQA (Castillo-Fraire et al., 2022, 2019), with better reproducibility of the electrochemical method. Both the electrochemical and enzymatic oxidation of CA led to the formation of several oxidized molecules. The formation pathway is suggested to involve the formation of caffeoyl-ortho-quinone, followed by the formation of caffeicin-like dimers. The other peaks detected correspond to compounds that are more polar than CA and are characterized by higher molecular weights. Further work is needed to isolate and characterize these oxidized molecules using semi-preparative HPLC or CPC and NMR, respectively. Further studies are encouraged to establish the biological activity of these oxidized compounds, either individually after isolation or directly as an oxidized solution. Finally, in future investigations, it would be interesting to use an on-line electrochemistry-LC-UV-Vis-ESI-MS 2 apparatus allowing the in-situ observation of less stable oxidized compounds than those we have identified. Because most polyphenols have very low bioavailability, their role in humans has been questioned. This paradox between low bioavailability and high bioactivity was resolved when scientists looked insight what happens when they reach the colon (Luca et al., 2020). Interestingly, the gut microbiota biotransforms them, producing low-weight metabolites that are better absorbed and may have different biological activities, which are often superior to of those their parents and have favorable residence time in humans (Carecho et al., 2020; González-Sarrías et al., 2017b). Thus, nowadays, new knowledge is focused on the biological effects of the gut-derived metabolites of polyphenols, making them a promising approach to prevent and attenuate neurodegenerative diseases (Carregosa et al., 2019b; Zhou et al., 2020). Therefore, there is great interest in studying the interaction of polyphenols with the gut microbiota. Indeed, identifying the specific bacteria responsible for the biotransformation of polyphenols into bioactive compounds, is crucial for understanding the impact of polyphenols and their metabolites on human health (Pasinetti et al., 2018b). Moreover, these findings can contribute to the development of a potential nutraceutical strategy that may beneficially regulate the gut-brain axis, which has a positive impact on human health (Cortés-Martín et al., 2020; Frolinger and Pasinetti, 2019; Westfall and Pasinetti, 2019). Firstly, we have investigated the biotransformation of 5-CQA using the resting cell method, by three gut bacteria species in monoculture, L. reuteri. B. fragilis and B. longum, which are representative of the dominated bacterial phyla in the gut. LC/MS 2 analysis of the biotransformed extracts revealed that only L. reuteri had the ability to biotransform 5-CQA into various metabolites related to the presence of cinammoyl esterase in its enzymatic machinery. Nevertheless, no metabolites were formed after biotransformation by B. fragilis and B. longum, indicating the absence of this enzyme in these bacterial species. Molecular networking analysis of 5-O-caffeoylquinic acid (5-CQA) biotransformation by L. reuteri highlighted for the first time its bioconversion to esculetin by an oxidative pathway, as well as other well-known gut-derived 5-CQA metabolites. The first step in the biosynthesis of this coumarin involves cinnamoyl esterase activity

Figure 59 .

 59 Figure 59. Aspect on the Colombia agar plate of L. reuteri (A) B. fragilis (B) and colonies after 48 h incubation at 37°C under anaerobic condition

threshold = 0. 4 ,

 4 minimum matched peaks = 3, precursor and fragment mass tolerance = 0.3, top hits kept = 3. The network was opened and visualized using Cytoscape (3.8.0). IV.C.6. Determination of calibration curves for 5-CQA, CA, esculetin by LC-MS/MS Standard solutions of 5-CQA, CA and esculetin at 1 Mm were prepared in MeOH and were further diluted to obtain working standard solutions at several concentration levels, in the range of 0.6-100 µg/ mL. Then, the calibration curve was obtained in the concentration range of 2.5-50 µg/mL.IV.D. Supplementation studiesThe strains were maintained on Columbia 3 agar plates. With the aim to evaluate the impact of 5-CQA in growth and/or metabolism of B. fragilis and L. reuteri, the experiments were carried out in presence of 5-CQA at different concentrations: 1 mM, 50 µM, 2.5 µM and 0.1 µM. An aliquot of 29 mL initial appropriate inoculum of each bacteria ~ 10 8 CFUmL -1 , OD600nm= 0.1 for B. fragilis and ~ 10 7 CFUmL -1 OD600nm= 0,7 for L. reuteri were established by suspending fresh bacterial isolation colonies In addition, a stock solution of 5-CQA (2 mM) was prepared in PBS at pH 5.8. Then, from this stock solution, adequate dilutions were done and then 1 mL of each was added at in 29 mL of each working culture. At each time, a control containing only bacteria (without 5-CQA) was incubated with all samples.The kinetic bacterial growth curve was established by absorbance measurementOD600nm and appreciated by bacterial enumeration expressed in CFU.mL -1 at different time (T0h, T6h, T24h and T30h). The incubation of cultures was carried out for overnight at 37°C in anaerobic chamber.At each sampling time, a count is carried out: this was done by cascade dilutions to 1:10 of the sample, in sterile water; indeed, 100 µL of each culture were taken and then diluted 10 times and plated on Columbia 3 agar for bacterial enumeration. Therefore, 100 μL of 10 -4 , 10 - for approximately 72 hours in anaerobic chamber or under anaerobic condition using sealed jars in presence of the anaerobic sachets (AnaeroGen AN0025) which absorbs rapidly all the oxygen in the jar and simultaneously generates carbon dioxide (Figure60) at 37 ° C.

Figure 60 .

 60 Figure 60. Jars with anaerobic sachet for inoculated petri dish incubation

  study the bacterial metabolites by evaluation of the metabolic profile after extraction. Each sample has been afterwards analysed by gas chromatography -mass spectrometry (GC-MS) for (SCFAs) and branched chain fatty acids (BCFAs).IV.D.2. Analysis of SCFAs, BCFAs,IV.D.2.1. Extraction methodsThe extraction of SCFAs, BCFAs, and AA was performed in a chemical fume hood from 1 mL of bacterial cell-free supernatants obtained after treatment with different 5-CQA concentrations and in each sampling time for both bacteria. The supernatants were mixed with, 1/100 methanol (80%) and 1/100 acetonitrile (5%), and 1/100 of 2 mg.mL -1 of diethylacetic acid (= 2-ethylbutyric acid) used as an internal standard (IS), then centrifuged for 5 min at 4000 rpm in 4°C in order to precipitate proteins then filtered with 0.2 μm filter before being stored in glass vials at -80 °C for further analysis. Similarly, a solution containing only BHI, extraction solvents and IS was used as a control.IV.D.2.2. Gas chromatography-mass spectrometry (GC-MS) analysis to determine SCFAs or BCFAs production a) GC-MS analysis Firstly, we have optimized and developed different metabolites extraction methods to have the maximum yield of these metabolites. Chromatographic conditions were tested (column type, composition of the mobile phases, pressure level, temperature, and elution gradient) to achieve a maximum of compounds separation quality and purity of chromatograms profile.The quantification and identification of these metabolites was carried out using a7020A-5975N GC-MS system (Agilent Technologies, Inc., Palo Alto, CA, USA) Data were collected with Enhance ChemStation G1701DA (Agilent software). Briefly, 1 μL of each extracted sample were directly injected in a ZB-WAX capillary column (30 mm length by 250 μm diameter with 0.25 μm thicknesses) using helium as a gas carrier with a constant flow rate of 1.5 mL/min. The temperature of the injector was kept at 220°C, and the injection was performed in splitless mode. The following oven temperature protocol was used as follows: an initial oven temperature of 50°C,5°C/min up to 180°C, 1 min at 180°C, and 20°C/min up to 220 °C for cleaning the column. The column was directly connected to the mass spectrometer which was operated at 70 eV and 260°C in the electron impact ionization mode with a full scan from 25 to 220 m/z. SCFA were identified by comparing their mass spectra with those held in the NIST MS Data Centre and 2HP-Wiley 138 library (Agilent) and by comparing their retention times with those of corresponding standards (Volatile Free fatty acid mixture CRM46975) purchased from Sigma. The peaks were quantified as the relative total ionic count abundance with respect to the internal standard.IV.E. Intestinal cell studyIV.E.1. Cell lines and growth cultureHuman NCI-H716 cells line, derived from a poorly differentiated adenocarcinoma of human cecum[START_REF] Park | Characteristics of cell lines established from human colorectal carcinoma[END_REF], was purchased from ATCC (LGC Standards, Molsheim, France). Cells were maintained and grown in suspension in Roswell Park Memorial Institute medium (RPMI-1640, Gibco, Life Technologies) supplemented with 10 % of fetal bovine serum (FBS), 1% of L-glutamine, 1% of 100 UI/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% of CO₂. Cell adhesion and differentiation into endocrine cells were initiated by growing cells for 48 h in 24-well culture plate coated with Matrigel Matrix (Corning®) (Gagnon and Brubaker, 2015);[START_REF] Reimer | A Human Cellular Model for Studying the Regulation of Glucagon-Like Peptide-1 Secretion[END_REF]. Caco-2 cells, obtained from American Type Culture Collection (ATCC), were cultivated in complete medium consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum, 1% L-glutamine and 1% penicillin and streptomycin. HT29-MTX cells were kindly provided by CRB CelluloNet (SFR Biosciences, CNRS UMS 3444, Inserm US 8, Université Claude-Bernard, Lyon, France) and were grown in the same medium as Caco-2 with only 10% of fetal bovine serum under a 5% CO2 water saturated atmosphere[START_REF] Lesuffleur | Increased growth adaptability to 5-fluorouracil and methotrexate of HT-29 subpopulations selected for their commitment to differentiation[END_REF]. The Raji B (ECACC 85011429), issued from human Burkitt's lymphoma cell-line, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine and 1% penicillin and streptomycin, at 37 °C in a 5% CO2 water saturated atmosphere.IV.E.2. Cellular model composed of four cells (quadricellular model)Human NCI-H716 cells were added to apical chamber of polycarbonate Transwell® inserts containing 25µg/cm² of matrigel and incubated at 37°C during 48h to obtain endocrine differentiated NCI-H716 cells. Upon confluence, Caco-2 and HT29-MTX cells were harvested with trypsin-EDTA and a predetermined amount of cells of each type were mixed prior to seeding to yield cell ratio of 8:1(Caco-2:HT29-MTX) on this insert containing endocrine differentiated NCI-H716 cells. After 14 days of culture, Raji B cells are added to the basolateral chamber to induce the differentiation of Caco-2 cells into M solution: INT and sodium lactate) has been prepared before. 100 µL of solution 1 and 5 mL of solution 2 were mixed. 100 µL of this mix was added on the 20 µL of each apical middle. 96

  Titre : L' acide 5-O-caféoylquinique, un polyphénol alimentaire, et ses métabolites produits par des bactéries intestinales : études électrochimiques, analyses LC-MS/MS, biotransformation et études biologiques Mots clés : polyphénol, microbiote intestinal, biotransformation, LC-MS/MS, électrochimie, intégrité de la barrière intestinale

  Les polyphénols sont connus pour leurs propriétés antioxydantes et comme modulateurs des voies de signalisation redox, expliquant en partie leur effet positif sur la santé humaine(Zhang and Tsao, 2016). Comme la plupart des polyphénols ont une biodisponibilité très faible, leur rôle chez l'homme a été remis en question. Ce paradoxe entre faible biodisponibilité et forte bioactivité a été résolu lorsque les scientifiques ont examiné ce qui se passe lorsqu'ils atteignent le colon(Luca et al., 2020). Il est intéressant de noter que le microbiote intestinal les biotransforme, produisant des métabolites de faible poids moléculaire qui sont mieux absorbés. Ces derniers peuvent avoir des activités biologiques différentes, souvent supérieures à leurs précurseurs, et ont un temps de demie-vie supérieur chez l'homme (Carecho et al., 2020; González-Sarrías et al., 2017). Actuellement, de nouvelles études se concentrent sur les effets biologiques des métabolites de polyphénols dérivés de l'intestin, ce qui en fait une approche prometteuse pour prévenir et atténuer les maladies neurodégénératives (Carregosa et al., 2019; Zhou et al., 2020). Par conséquent, l'étude de l'interaction des polyphénols avec le microbiote intestinal suscite un grand intérêt. En effet, identifier les bactéries spécifiques responsables de la biotransformation des polyphénols en composés bioactifs est crucial pour comprendre l'impact des polyphénols et de leurs métabolites sur la santé humaine (Pasinetti et al., 2018). De plus, ces résultats peuvent contribuer au développement d'une stratégie nutraceutique d'intérêt susceptible de réguler de manière bénéfique l'axe intestin-cerveau, et ainsi avoir un impact positif sur la santé humaine (Cortés-Martín et al., 2020; Frolinger and Pasinetti, 2019; Westfall and Pasinetti, 2019). Parmi les polyphénols alimentaires, l'acide 5-O-caféoylquinique (5-CQA), également connu sous le nom d'acide chlorogénique, a montré des avantages potentiels pour la santé humaine. Dans ce contexte, nous avons soumis le 5-CQA à une étude de biotransformation en utilisant Lactobacillus reuteri, Bacteroides fragilis et Bifidobacterium longum par la méthode de "resting cells". Ces bactéries ont été sélectionnées en fonction de leur appartenance aux principaux phyla présents dans le microbiote intestinal humain. Sur la base de la courbe de croissance de ces bactéries obtenue par la mesure de la densité otpique à 620 nm, la réaction de biotransformation a été réalisée pendant 24 h lorsque la phase stationnaire était atteinte. Le tampon phosphate (PBS) a été choisi pour solubiliser le 5-CQA et son métabolite l'acide caféique (CA) car ce tampona été décrit précédemment comme ayant des caractéristiques proches de la composition bactérienne intracytoplasmique (Mortelé et al., 2019). L'étape de biotransformation a été réalisée à pH 5,8, proche du pH mesuré dans le côlon. Le 5-CQA a été dissous dans du PBS à 1 mM, une concentration proche de celles mesurées dans le côlon après l'ingestion de café ou de fruits (Clifford, 1999). Après centrifugation, le culot et le surnageant ont été traités séparément à l'acétate d'éthyle pour obtenir deux extraits qui ont été analysés par LC-UV-ESI-MS 2 en mode négatif. Alors que L. reuteri était capable de biotransformer le 5-CQA, aucun métabolite biotransformé n'a été détecté après bioconversion par B. fragilis et B. longum. Pour valider ces résultats, nous avons recherché in silico, dans le génome de ces bactéries, l'enzyme responsable de la biotransformation du 5-CQA, nommée cinnamoyl esterase ou feruloyl esterase (EC.3.1.1.73), qui a déjà été trouvée chez L. johnsonii (accession number NC_005362) (Bel-Rhlid et al., 2013). Une comparaison génomique entre le gène déjà décrit chez L. jonhsonii et ceux présents dans notre souche L. reuteri DSM 20016 (GCA_001436151 a montré 71 % de similarité avec le gène Locus Tag nommé LREU_RS08790. Néanmoins, ce gène n'a pas été trouvé dans le génome de B. fragilis et B. longum, ce qui suggère que les résultats de biotransformation obtenus par LC-ES-MS 2 sont conformes aux analyses in silico. La recherche sur Pfam a mis en évidence la présence du même domaine, hydrolase_4 serine aminopeptidase S33 dans ces deux gènes qui appartiennent à la famille de Alpha/Beta hydrolases (El-Gebali et al., 2019). Ce domaine catalytique se retrouve dans une très large gamme d'enzymes comme les estérases. Cette cinnamoyl estérase a permis l'hydrolyse du 5-CQA en CA et en acide quinique. Le CA a effectivement été détecté dans le chromatogramme obtenu par LC-ESI-MS 2 . Par contre,l'acide quinique, étant trop polaire, n'a pas été extrait par l'acétate d'éthyle ou n'a pas été retenu sur la colonne C-18 utilisée. Les données LC-MS/MS de l'extrait de L. reuteri ont ensuite été traitées par le logiciel MZMine 2.53 et par Python v3.7 matchms v0.9.2, pour une analyse en réseau moléculaire où chaque spectre de masse en tandem est décrit comme un noeud et les alignements entre spectres comme des liens définis par un score de cosinus (cos) (http://gnps.ucsd.edu). Un cos élevé signifie une grande similarité entre deux spectres MS/MS. Le réseau moléculaire (MN) créé a mis en évidence 110 noeuds. Un cluster englobait la majorité des noeuds connectés et 45 noeuds correspondaient à des "auto-boucles", ce qui représentent une caractéristique spectral unique. Des métabolites spécifiques de cette biotransformation sont apparus dans tous les clusters, mais principalement sous forme de noeuds "auto-bouclés". Nous avons ensuite annoté les MN par déréplication en utilisant la bibliothèque GNPS. Parmi les métabolites issus de la bioconversion du 5-CQA par L. reuteri, le cis-CA, le trans-CA, l'acide coumaroyl quinique et l'esculetine ont attiré notre attention en raison de leur structure proche du 5-CQA. Pour valider certaines de ces annotations, divers témoins analytiques ont été injectés et ont conduit à l'identification du 5-CQA, de l'esculetine et du trans-CA. De manière surprenante, les isomères géométriques cis et trans du CA ont été identifiés dans le MN. À notre connaissance, il s'agit de la première indication de la présence de ces deux isomères de CA après biotransformation du 5-CQA par des bactéries ou par le microbiote intestinal humain. L'analyse du réseau moléculaire de la biotransformation du 5-CQA par L. reuteri a mis en évidence pour la première fois sa bioconversion en esculetine par une voie oxydative. Dautres métabolites de 5-CQA bien connus ont également été identifiés. Nous avons proposé une voie de biosynthèse de l'esculétine à partir du 5-CQA. La première étape de la biosynthèse de cette coumarine implique une activité de type cinnamoyl estérase qui hydrolyse la liaison ester entre les acides caféique et quinique. Ensuite, le cis-CA est formé par une isomérase présente dans la souche de L. reuteri étudiée, puis il peut être converti par une oxydase en caféoyl-o-quinone. Finalement, cette dernière est stabilisée en esculetine. Ainsi, ces résultats donnent un aperçu de la voie de biotransformation d'un composé naturel, le 5-CQA, qui est fortement ingéré par l'alimentation. Il est à noter que l'esculetine est une molécule de grand intérêt pour la recherche médicale (Garg et al., 2020). Elle a ainsi montré certaines activités biologiques telles que l'inhibition du stress oxydatif et de l'inflammation ; elle est également connue pour posséder des propriétés anticancéreuses (Zhang et al., 2022). Pour mieux comprendre la voie de biotransformation oxydative menant à l'esculetine, nous avons étudié l'oxydation du 5-CQA et du CA par voie électrochimique et par voie enzymatique. Dans un premier temps, le comportement électrochimique du 5-CQA et du CA a été étudié par voltampérométrie cyclique dans du PBS 0,1 M à pH 5,8 dans des conditions similaires à celles des études de biotransformation. Comme attendu pour les deux composés, la fonction ortho-diphénol est oxydée selon un mécanisme à deux électrons-protons pour former une ortho-quinone (visible sur le balayage anodique à Epa = +0,55 V et à Epa = +0,38 V, respectivement pour le 5-CQA et le CA), qui est réduite en sa forme catéchol sur le balayage cathodique retour (Epc = + 0,11 V et Epc = + 0,14 V, respectivement pour le 5-CQA et le CA). Ensuite, l'électrolyse à potentiel contrôlé de 5-CQA et CA a été réalisée dans du PBS 0,1 M à pH 5,8 sur grille de platine dans une cellule spectrophotométrique UV-Visible. Dans un premier temps, les spectres UV-Visible ont été enregistrés en temps reel au cours du tracé de la voltampérométrie à balayage linéaire LSV à différentes valeurs de potentiel. Jusqu'à + 0.75 V et + 0.52 V respectivement, le 5-CQA et le CA sont restés sous leur forme native et la longueur d'onde d'absorption maximale λmax a été mesurée à 320 nm. Au dessus de ces valeurs de potentiels, une bande d'absorption était visible autour de 400 nm pour les deux composés, attestant de leur oxydation sous leur forme quinone. Dans un deuxième temps, la technique de Comme il existe une interaction entre les bactéries dans l'intestin, nous avons étudié, dans une autre partie, si ces interactions pouvaient influencer le processus de biotransformation. À cette fin, nous avons co-cultivé l'espèce L. reuteri qui est impliquée dans la biotransformation du 5-CQA avec B. fragilis, qui n'a pas montré la capacité de le biotransformer. Comme résultat préliminaire, la formation d'esculetine a été favorablement augmentée de 24 % par la co-culture de ces deux bactéries par rapport à L. reuteri seule. La prochaine étape consistera à soumettre le 5-CQA au processus de biotransformation impliquant les trois bactéries étudiées, L. reuteri, B. fragilis et B. longum. Une autre question qui doit être abordée est de savoir comment les polyphénols et leurs métabolites issus de l'intestin modulent également la composition et la fonction du microbiote intestinal, ce qui pourrait avoir un impact positif sur la santé humaine (Selma et al., 2020). Ainsi, nous avons étudié l'impact du 5-CQA sur la croissance et les profils métaboliques de L. reuteri et B. fragilis. Dans cette étude, l'impact de différentes concentrations de 5-CQA, à 0,1, 2,5, 50 et 1000 µM ont été évaluées sur les deux souches bactériennes intestinales L. reuteri et B. fragilis. Cette gamme de concentrations a été choisie de la concentration la plus faible aux concentrations physiologiques pouvant potentiellement atteindre l'intestin. Le 5-CQA a été ajouté au début de la culture puis les bactéries ont été incubées dans une chambre anaérobie pendant 30 heures à 37°C. Les surnageants de culture ont été recueillis au début (temps 0) et après 6, 24 et 30 heures d'incubation pour mesurer la DO à 620 nm reflétant la croissance bactérienne et pour déterminer la concentration bactérienne (CFU.mL -1 ). Des cultures des souches testées sans 5-CQA ont également été réalisées en tant que témoins, appelés L. reuteri non traité ou B. fragilis non traité. L'effet du 5-CQA sur la croissance de L. reuteri et de B. fragilis a été déterminé en comparant les paramètres de croissance des cultures traitées avec différentes concentrations de 5-CQA à ceux des cultures non traitées. Pour étudier l'impact de différentes concentrations de 5-CQA sur la production métabolique de ces deux espèces bactériennes, comme les acides gras à chaines courtes (SCFA) et les ramifies (BCFA), les surnageants des cultures cultivées dans des conditions standard (BHI = 25 μg.mL-1 d'hémine) ainsi que dans des concentrations de 5-CQA supérieures ont été analysées par GC-MS. Nous avons finalement observé que le 5-CQA n'avait aucun effet ni sur la croissance, ni sur les productions métaboliques (SCFAs et BFCAs) des deux souches bactériennes étudiées. L'intégrité et la fonction de la barrière intestinale sont essentielles au maintien de l'homéostasie intestinale, et donc de la santé de l'hôte (Singh et al., 2019). Cette barrière intestinale est une couche unique constituée de différents types de cellules telles que les entérocytes, les cellules de Goblet, les entéroendocrines, les cellules de Microfold (M) et de Paneth. Ces cellules sont scellées par des protéines transmembranaires de jonction serrée (TJ) (par exemple, occludine, claudines, molécules d'adhésion jonctionnelle et tricelluline) ainsi que par des protéines membranaires périphériques (par exemple, Zonula occludens (Zo-1) et cinguline). Leur rôle est d'empêcher la perméabilité paracellulaire et de maintenir la fonction de barrière intestinale. En outre, la couche de mucus empêche le contact direct entre les microbes intestinaux et les cellules épithéliales (Chelakkot et al., 2018; Hooper, 2015). La perturbation des jonctions serrées entraîne un dysfonctionnement de la barrière, suivi de la translocation des agents pathogènes et des toxines bactériennes au niveau systémique et de l'activation du système immunitaire et de l'inflammation, contribuant ainsi à plusieurs conditions pathologiques (Chelakkot et al., 2018). Des preuves issues d'études in vitro et animales suggèrent que les polyphénols peuvent réguler et maintenir l'intégrité et la fonction intestinales, par le biais de plusieurs mécanismes (Bernardi et al., 2020; Wan et al., 2021). La plupart des études in vitro ont été réalisées à l'aide de la lignée cellulaire Caco-2, puis T84 et HT29/B6 (Bernardi et al., 2020). La plupart des connaissances actuelles sur les mécanismes du microbiome proviennent de systèmes modèles tels que les systèmes in vitro, ex vivo et animaux (Galloway-Peña and Hanson, 2020). Bien que les modèles animaux soient fréquemment utilisés, la transposition des données à l'homme a été remise en question (Bjornson-Hooper et al., 2022; Desmoulin-Canselier and Moutaud, 2019; Pound and Ram, 2020; Seok et al., 2013). En outre, elles impliquent des problèmes éthiques et sont assez coûteuses (Fitzgerald et al., 2015). Par conséquent, les approches in vitro à base de cellules peuvent constituer une alternative à l'expérimentation animale (Bédard et al., 2020). La culture cellulaire permet ainsi aux scientifiques d'étudier les mécanismes cellulaires et moléculaires d'une manière relativement simple et reproductible (Joseph et al., 2018). Pour tenter de se rapprocher de l'intestin humain et éviter l'utilisation de modèles animaux, le Dr Latifa BOUSARGHIN et son équipe (NuMeCan), ont développé un modèle in vitro quadruple. Ce modèle original est composé d'entérocytes (Caco-2), de cellules de gobelets sécrétant du mucus (HT29-MTX), de cellules de micro-plis (cellules M) et de cellules entéroendocrines (NCI). Cinq types de cellules ont été utilisés pour ce qui suit, et doivent donc être

Table 1 .

 1 The main flavonoids and their food sources(Tomás-Barberán et al., 2020). ......................... 10

Table 2 .

 2 5-CQA content in Arabica vs Robusta, compared to total chlorogenic acids and effects of roasting time on this content[START_REF] Lu | Chlorogenic acid: A comprehensive review of the dietary sources, processing effects, bioavailability, beneficial properties, mechanisms of action, and future directions[END_REF] ...................................................................................... 19

Table 3 .

 3 LC-MS 2 analysis of electrochemical and enzymatic oxidized solution of 5-CQA. Green cercles indicate the presence of peak and red ones the absence of peak ........................................................... 82

Table 4 .

 4 LC-MS 2 analysis of electrochemical and enzymatic oxidized solution of CA: green cercles indicate the presence of peaks and red ones the absence of peaks. Starred values refer to ions different from the deprotonated molecular ion .................................................................................................... 88

Table 5

 5

. LC-DAD-ESI-MS 2 metabolites identified or detected in SN extract after 5-CQA biotransformation by L. reuteri. .......................................................................................................... 103

Table 6. Comparison of spectral patterns of esculetin formed using different methods ..................... 109

List of schemes

Scheme 1. Biosynthesis of 5-CQA acids and related phenolic compounds. Abbreviations: PEP (phosphoenolpyruvate), PAL (phenylalanine ammonia lyase), C4H (cinnamate 4'-hydroxylase), 4CL (4-cinnamoyl CoA ligase), HQT (hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase), C3H (p-coumaroyl-3'-hydroxylase), and CCoAMT (caffeoyl-CoA-3-O-methyltransferase). ............. 17

Scheme 2. Mechanisms of formation and structures of oxidized products derived from 5-CQA (adapted from (Castillo-Fraire et al., 2019) and (Castillo-Fraire et al., 2022) ..................................................... 85 Scheme 3. Mechanisms of formation and structures of some oxidized products derived from 5-CA (adapted from (Pati et al., 2006) ........................................................................................................... 89 Scheme 4. Putative biotransformation pathway of 5-CQA by L. reuteri. Black arrows: biotransformation already reported; blue arrows: putative mechanism of formation of the identified esculetin; hashed arrows: two possible pathways for ion at m/z 135. Compounds marked in bold were identified using analytical standards or by MN and in italic were detected and putatively identified by comparison data with previous results. ............................................................................................... 105

Johnson et al., 2019; Raffaelli et al., 2002).

  Stilbenes are less present in the human diet. Resveratrol the main phenolic compound characteristic of this group, is found in grapes and red wine. It generally occurs in glycosylated forms rather than as cis/trans isomers, as well as in oligomers containing different resveratrol units, such as δ-viniferin and 𝜀-viniferin[START_REF] Shen | Natural stilbenes: an overview[END_REF].

Vuolo et al., 2019; Zhang and Tsao, 2016).

• , a less harmful radical, which is firstly stabilized by resonance before being involved in non-toxic reactions (

Lu et al., 2020). I.B. The human gut microbiota

	revealed that 5-CQA and its
	metabolites were safe to use and could have beneficial effects as a pharmaceutical, by in vitro
	and ex vivo profiling assays, according to ICH S7A guidelines (Amano et al., 2019). However,
	studies suggest that further research should focus on 5-CQA, including its absorption and
	bioavailability in humans, interactions with microorganisms and evaluation of the benefits of
	5-CQA and its metabolites (I.B.1.The Microbiome: An overview
	Trillions of microorganisms, including bacteria, viruses, archaea and eukaryotes live in
	the human body (

Thursby and Juge, 2017). These

	microbial organisms collectively referred
	to as microbiota, can be found in different body parts, including the skin, oral cavity, intestinal
	tract, urinary tract, respiratory tract, vagina (

Lloyd-Price et al., 2016). Indeed, the term microbiota is followed by the location of the specific environment referred to; for example, the gut microbiota represents the entire collection of microorganisms in the gastrointestinal tract (Marchesi and Ravel, 2015). On the other hand, in the genomic context, microbiome refers to

	the all the genetic material (collective genomes) of the micro-organisms inhabiting a particular
	environment (

Valdes et al., 2018). Thus

	, the microbiome represents the microbiota and their
	"theatre of activity" by which it includes microbial structures, metabolites, genetic material,
	and surrounding environment (

Berg et al., 2020). Human

	microbiome research has become a
	topic of great interest among scientific community, medical research, and industry (Proctor,

(Sender et al., 2016). Nonetheless, viral, fungal and archaeal communities, have recently aroused growing interest, since their dynamic presence impacts gut bacteria, and directly the host well-being (Matijašić et al., 2020).

	(∼10 times more), representing the highest number of microorganisms present in the human
	body. However, Sender et al., found that this ratio is closer, and the number of bacteria in
	humans is similar to the number of host cells. It has been estimated that, in humans, the total
	number of bacteria in a person with a body weight of 70 kg is around 3.0 × 10 13 II.B.1. 2. Gut microbiota: Structure and diversity
	Of all microorganisms that live in the human body, most of them are found in the
	intestine, and the gut encodes more than 3 million genes (150 times more than human genes).
	Thus, it is considered as a reservoir of genes in the human body (

Qin et al., 2010; Tierney et al., 2019). The

  gut microbiota is remarkably rich and diverse, predominantly by bacteria,

	classified taxonomically according to phylum, class, order, family, genus and species
	(Rinninella

et al., 2019b). The

  highest amount of bacteria live within the colon, reaching a density of 10 12 bacteria per gram, with over 1000 species (

Dieterich et al., 2018; Lozupone et al., 2012).

	In regard to the microbiota, it is well known the tremendous interindividual variation
	and it has been established that microbiota profile is unique to each person (Huttenhower et
	al., 2012). Nevertheless, it is recognized that Firmicutes, Bacteroidetes, Actinobacteria,
	Proteobacteria and Tenericutes, are dominant phyla of human gut microbiota
	whereas Fusobacteria,	Saccharibacteria,	Spirochaetes,	Synergistetes	and
	Verrucomicrobia phyla are found in lower abundance (

Almeida et al., 2019).

  Bacillus spp and Clostridium spp, Enterococcus spp and Ruminococcus spp.,

	Firmicutes represents the most diverse and predominant phylum in the human gut,
	with more than 200 different Gram-positive bacteria; Lactobacillus spp, Mycoplasma spp,

among them (Rajilić-Stojanović and de Vos, 2014). Within

  the gut, these genera include microbial species that are considered the key players in gut homeostasis such as Lactobacillus reuteri,

	Faecalibacterium prausnitzii, Roseburia intestinalis,

and Clostridium spp (Britton, 2017; He et al., 2021; Tamanai-Shacoori et al., 2017).

	Bacteroidetes are Gram negative bacteria, which together with Firmicutes, represent
	the two predominant phyla in the healthy gut (more than 90%)

(Eckburg et al., 2005). Bacteroides, Alistipes, Parabacteroides and Prevotella are the representative genera of Bacteroidetes (Gibiino et al., 2018). These are followed by Actinobacteria encompassing

	Gram-positive species, consisting of less than the 10% of total bacteria and mainly represented
	by Bifidobacterium ( e.g B. adolescents, B. animalis, B. breve, B. bifidum, B. longum and B.
	angulatum) (

Binda et al., 2018). Proteobacteria

	, encompasses Gram-negative bacteria,
	including notable pathogens (e.g. Escherichia, Helicobacter, Salmonella, Vibrio) (Rizzatti et
	al., 2017). Verrucomicrobia are Gram negative bacteria, represented by Akkermansia
	muciniphila, the only known species of this phylum. A. muciniphila colonizes the mucus layer
	of the gut epithelium, consisting 3-5 % of the total microbiota community, and with a great
	interest related to host health and diseases (

Xu et al., 2020).

	Among non-bacterial communities in the gut, viruses are the most abundant, mainly
	corresponding to the order Caudovirales and Microviridae families. The human gut also
	harbours fungi such as the genera Candida, Penicillium, Saccharomyces, Aspergillus,
	Cryptococcus, Malassezia etc. Furthermore, Methanobrevibacter smithii, Methanosphaera
	stadtmanae, Methanomassiliicoccus luminyensis are the members of the archeal community
	identified in the human gut (

Matijašić et al., 2020).

	II.B.1.3. The lifetime journey of human gut microbiota
	a) Early life
	The gut microbiota is a dynamic ecosystem, where development and maturation
	processes continue during several stages of life including infancy, adolescence, adulthood, and
	aging.
	Over the years, the colonization process of the gut microbiota has been a topic of debate
	among academia (

Blaser et al., 2021). Some

	scientists have suggested that colonization
	primarily may come through the placenta and in utero after detection of microbes and traces of
	bacterial DNA in the placenta, amniotic fluid and meconium (

Aagaard et al., 2014; Collado et al., 2016; Jiménez et al., 2008). This hypothesis is questionable by others suggesting that these findings could be a result of contamination (Lauder et al., 2016).

	Despite controversy, there is a consensus among the majority of scientists that humans
	are born sterile, and microbial colonization of the gut begins at birth. When birth is vaginally,
	a vertical transfer of bacteria from maternal genital and skin flora to infants through the birth
	canal initiates habitation, predominantly by Lactobacillus, Prevotella and Sneathia spp.

(Dominguez-Bello et al., 2010; Korpela, 2021). After

	birth, the infant`s gut is colonized by
	maternal gut sources (

Ferretti et al., 2018) and

	and hospital environment, they acquire Staphylococcus, Corynebacterium, and
	Propionibacterium spp.
	is mainly dominated by facultative anaerobes,
	including Firmicutes (Staphylococcus, Streptococcus), Proteobacteria (Enterobacteria) and
	anaerobic species such as Bifidobacterium, Bacteroides, and Clostridium (Korpela et al.,
	2018). However, since Caesarean section (C-section) infants are exposed firstly to the skin

(Dominguez-Bello et al., 2010).

	After birth, the microbiota composition of infants shows a low of diversity (Milani et
	al., 2017). Scientists consider that by the age of 1-3 years old, the bacterial diversity of the
	infant's gut microbiota gradually increases (

Derrien et al., 2019).

	However, the gut microbiota of infants and children is highly affected by different
	factors such as delivery mode, mode of feeding (breastfeeding versus formula), antibiotics,
	environment, and stress (

Bokulich et al., 2016; Callaghan et al., 2020).

	The delivery mode is the main factor affecting the gut microbiota composition at the
	earliest stage of life (1 months-1 year)

(Shao et al., 2019). The

  gut microbiota of infants delivered by C-section is less diverse compared to vaginally born infants with low levels of Bifidobacterium and traces of Bacteroides species and an increase of pathogenic bacteria

Loughman et al., 2020; Sarkar et al., 2021; Skillington et al., 2021; H. Zhou et al., 2021).

	In addition to the delivery mode, other factors may affect gut microbiota, including diet
	(breastfeeding vs. formula feeding), physical illness, antibiotic use, and stress, which in turn
	may alter microbiota profile and consequently negatively influence long-term health (Cryan
	et al., 2019b). Emerging evidence suggests that imbalance (dysbiosis) of gut microbiota in
	early-life, is associated with upcoming diseases such as allergies, metabolic diseases (obesity,
	diabetes mellitus, type 1, cardiovascular diseases) and neuropsychiatric disorders, even with
	behavioural patterns (The importance of early life microbiota composition for shaping future health, with
	particular attention to brain development, is an emerging topic in the neuroscience field. Thus,
	the scientists are considering that taking care of and shaping the gut microbiota at early life
	stage may favorably affect health in the years to come (

S. Wang et al., 2018).

	b) Adulthood
	However, even in pre-adolescents (children 7-12 years old) and adolescents, the
	microbiota does not reach adult microbiota profile maturation, including functional and
	taxonomic patterns (

Agans et al., 2011; Hollister et al., 2015).

	In adults, the gut microbiota reaches maturity and has long-term stability (

Faith et al., 2013). Although is largely affected by infections, antibiotics, and diet it maintains its stable equilibrium state through the known resilience phenomena that characterize the gut microbiota (Sommer et al., 2017).

	c) Aging
	Studies have shown that aging is associated with the alternation of gut microbiota
	composition characterized by the decrease of diversity, particularly the reduction of short chain
	fatty acids (SCFA) producing species and an increase of opportunistic species (

Bana and Cabreiro, 2019; Ragonnaud and Biragyn, 2021). In

  a cross-sectional centenarian study, a decrease in anaerobic bacteria such as Bifidobacterium spp. and an increase in Clostridium and Proteobacteria, were observed among Japanese people (

Odamaki et al., 2016). A

	study
	exploring age-related differences showed that the microbiota of centenarians was associated
	with a decrease in Faecalibacterium prausnitzii and an increase of pathobionts (Biagi et al.,
	2010). Given the roles of Bifidobacterium spp. and Faecalibacterium prausnitzii in the immune
	system and metabolic processes, these decreases may explain the low systemic inflammatory
	status and malnutrition in older adults (

Guigoz et al., 2008).

	Moreover, altered gut microbiota, caused by aging, contributes to several dysfunctions
	of the gut, including disruption of barrier integrity, which leads to `leaky gut `syndrome and
	translocation of pathogenic bacteria and endotoxins into the circulatory system which induces
	an inflammatory response resulting in the increased risk of brain and heart diseases (

Cryan et al., 2019a; Ragonnaud and Biragyn, 2021). Hence

	, the gut microbiota is considered a key
	player to healthy ageing (

Claesson et al., 2012; Ragonnaud and Biragyn, 2021). A

	cohort
	study conducted by Wilmanski et al. identified that the microbiome pattern reflects healthy
	aging, characterized by depletion of Bacteroides or high gut microbiome diversity (Wilmanski
	et al., 2021). Therefore, scientists have questioned whether taking care of our microbiota might
	be linked to longevity (

Biagi et al., 2016; Y. Zhou et al., 2021) and beyond that, whether the fountain of youth lies in the gut microbiome (Cryan et al., 2019a).

	I.B.2. Factors shaping gut microbiota
	In addition to delivery mode, it is well-known that both genetic and environmental
	factors contribute to microbiome composition and function (

Benson et al., 2010; Spor et al., 2011; Xie et al., 2016). However

	, it has been established that the environment (diet, lifestyle,
	and medications) can overcome the heritage (

Qin et al., 2020; Rothschild et al., 2018; Scepanovic et al., 2019).

	I.B.2.1. Genetics
	Evidence obtained from mouse models suggests that host genetic contribute to shaping
	the gut microbiota (

Kemis et al., 2019; Suzuki et al., 2019; Ussar et al., 2015). Twin

	studies
	have provided further insight and assessment of the role of genetics in determining e the
	characteristics of the microbiome profile (

Goodrich et al., 2014; Turnbaugh et al., 2009; Xie et al., 2016). Several

  gut microbial taxa have been found to be heritable in humans, and

	Christensenellaceae (belonging to the phylum Firmicutes) showed the highest heritability
	followed by Archea (Goodrich et al., 2014). The same team of researchers, following a study
	with a larger sample size, detected a link between heritable bacteria and human genes related
	to diet, metabolism and immunity (

Goodrich et al., 2016).

	I.B.2.2. Diet
	Dietary patterns are one of the main contributing factors that shape the gut, and
	consequently affect health (

David et al., 2014; Moles and Otaegui, 2020; Sandhu et al., 2017) and even mental health (Firth et al., 2019; Taylor et al., 2020; Watson, 2020a).

	There is evidence that some popular diets, such as Mediterranean, vegetarian, vegan,
	Western, and gluten-free diets are associated with differences in the gut microbiota

(Rinninella et al., 2019a). The intake of such known Mediterranean diet, which is rich in fruits, vegetables, coffee, wine, and olive oil fish, cereals, nuts, seeds and aromatic herbs, influences the gut microbiota (Merra et al., 2020). For example, consumption of the Mediterranean diet for one

	year by obese men (n = 20) significantly decreased the genera Prevotella and increased the
	Roseburia genus and F. prausnitzii,

(Haro et al., 2016). Another

	study carried out in a cohort
	of Italian individuals (n = 153) reported that adherence to the Mediterranean diet was associated
	with increased levels of faecal (SCFAs) which correlated with the enrichment of Firmicutes
	and Bacteroidetes (

De Filippis et al., 2016). Likewise

	, a study of a Spanish cohort (n = 150)
	also observed association of the Mediterranean diet with higher levels of butyrate-producing
	taxa such as Roseburia faecis, Ruminococcus bromii and Oscillospira (Flavonifractor) plautii

(Rosés et al., 2021). Garcia

  -Mantrana et al. highlighted the positive impact of this diet on the gut microbiota, particularly by enhancing the abundance of Bifidobacteria and levels of SCFAs

(Garcia-Mantrana et al., 2018).

	Moreover, emerging evidence suggests that the Mediterranean diet is associated with
	higher cognitive performance and a reduced incidence of brain diseases (

Fresán et al., 2019; Karstens et al., 2019).

	A randomized controlled trial that evaluated dietary intervention in
	major depression (SMILES) confirmed that a poor diet was correlated with depression
	symptomatology and showed that the Mediterranean diet intervention improved depression
	scores (Jacka et al., 2017). The compounds present in the Mediterranean diet, mainly phenolic
	compounds, indicate that apart from their antioxidant effects, they can positively impact the
	human health by modulating the gut microbiota (

Bucciantini et al., 2021; Selma et al., 2020).

	On the other hand, consumption of a Western diet is characterized of high intake of
	processed high-fat meat, saturated fats, refined grains, sugar, salt, alcohol and corn-derived
	fructose, fast food, including French fries, and soft drinks. Recently, scientists have
	investigated the effects of long-term dietary patterns and specific nutrients on the composition
	and activity of gut microbiota. They found that the Western diet was accompanied by a high
	abundance of inflammatory bacteria, namely, Clostridium bolteae, Ruminococcus obeum, R.
	gnavus and Blautia hydrogenotrophica and a decrease in commensal bacteria such as
	Firmicutes (Roseburia, Eubacterium rectale and Ruminococcus bromii)

(Bolte et al., 2021).

	Moreover, clinical studies support the hypothesis that a diet consisting of animal and processed
	foods, sugar and alcohol, by affecting the gut microbiota, may trigger obesity, metabolic
	diseases, cognitive

impairment and increased anxiety (Attuquayefio et al., 2017; Peris- Sampedro et al., 2019; Zinöcker and Lindseth, 2018).

(Stillhart et al., 2020). Once

	in the
	colon, a complex reciprocal interaction occurs between drugs and resident gut microbes

(Weersma et al., 2020).

	Several in vitro and in vivo studies in humans have shown that some drugs are
	associated with changes in gut microbiota, including decreased diversity and taxonomic
	changes (

Forslund et al., 2015; Jackson et al., 2016). For

	example, when scientists screened
	100 common drugs prescribed for various diseases, they found that a quarter of them had the
	antibiotic effects on 40 bacterial strains (

Maier et al., 2018). Indeed

	, over the years, the
	strongest evidence of drug effects on the microbiota comes from human cohort studies. A
	recent investigation using metagenomic sequencing, found that 19 of 41 drugs were associated
	with the gut microbiome (

Vila et al., 2020). A

	broad range of drugs that affect the gut
	microbiota, have been identified, including proton pump inhibitors (PPIs), metformin, statins,
	osmotic laxatives, inflammatory bowel disease (IBD) medications, female sex hormones,
	benzodiazepines, antidepressants,

and antihistamines (Falony et al., 2016; Zhernakova et al., 2016). Furthermore, scientists have found that the use of multiple drugs (polypharmacy) can alter the gut microbiota differently from single drug administration (Rogers and Aronoff, 2016), with a significant negative correlation (Ticinesi et al., 2017).

	Scientific data have revealed a complex dynamic interaction between drugs and the gut
	microbiota. As drugs affect the gut microbiota, the gut microbiota can also affect the
	pharmacokinetic profiles of drugs and consequently their efficacy and toxicity (

Weersma et al., 2020; Zhang et al., 2018). The

	gut microbiota can extensively transform drugs using
	microbiome-derived enzymes (

Koppel et al., 2017; Zimmermann et al., 2019). The

	drugs biotransformation's include several chemical reactions and mainly correspond to the
	first-phase reactions of reduction and hydrolysis. The enzymatic machinery of gut microbiota
	may contribute to the activation of prodrugs (e.g., sulfasalazine and prontosil, as shown in
	Figure 9), drug inactivation (e.g., L-Dopa, digoxin and methotrexate), and an increase in drug
	toxicity (e.g., sorivudine and brivudine) (
	microbial

Spanogiannopoulos et al., 2016; Zhang and Chu, 2021; Zimmermann et al., 2021).

			O	O
	O			S	NH	N	O		O	S O
	O H	N	N	Metabolic reduction	O H	NH 2	N H 2 +	NH	N
	O H			Gut microbiota	O H
	Sulfasalazine					5-aminosalicylic acid (5-ASA)		sulfapyridine
	(prodrug)					active drug

Di Meo et al., 2013).

•+ cation radical (

Table 6 .

 6 Comparison of spectral patterns of esculetin formed using different methods

(Caslin et al., 2021).

  Moreover, L. reuteri can produce various volatile metabolites with diverse biological activity such as 1,3-propanediol, detected in our samples, which is one of the oldest known fermentation products and has enormous applications in polymers, cosmetics, foods, adhesives, lubricants, laminates, solvents,

	antifreeze, and medicines industries (Baeza-Jiménez et al., 2011). L. reuteri is a promising
	probiotic bacterium. It has been found that metabolism of glycerol by L. reuteri can result in
	excretion of a metabolic intermediate, 3-hydroxypropionaldehyde known as reuterin, which
	has greater antimicrobial activity against potential pathogens such as Salmonella, Clostridium,
	and Escherichia species (Mu et al., 2018). Specifically, L. reuteri is able to bioconverse
	glycerol into 1,3-propanediol and 3-hydroxypropionaldehyde (Figure 51). As a first step,
	glycerol is transformed to 3-HPA (3 hydroxypropionaldehyde or reuterin) by glycerol
	dehydratase, which is further reduced to 1,3-propanediol by 1,3-propanediol oxidoreductase.
	This stage uses NADH and regenerates NAD + , which is considered a potential mechanism by
	which L. reuteri favors glycolysis by readjusting its redox status (Britton, 2017). Nonetheless,
	these intermediate metabolites were not detected under our experimental conditions.

Rinninella et al., 2019b). Besides

  health. Nevertheless, it should be emphasized that more research needs to be conducted, first to elucidate the complexity of the gut microbiota related to brain disease. It remains still unclear whether alteration of the gut microbiota plays an initial role in the pathogenesis and progression It is now known that regular consumption of dietary polyphenols can maintain or restore the balance of the gut microbiota, thus contributing to the general human well-being. Therefore, understanding the complex interactions between polyphenols and the microbiota is pivotal to promote the overall health benefits of dietary polyphenol consumption and, in addition, represents a promising strategy to identify a potential approach toward a modifiable gut microbiota, which could lead to positive effects on brain function.In addition, multiple pathways are involved between gut microbiota and brain crosstalk: neural (via vagus nerve, enteric nervous system and spinal nerves) immune (via cytokines and chemokines secretion), endocrine (via HPA axis, tryptophan, serotonin) and metabolic (due to short-chain fatty acids and microbial metabolites) pathways. However, the fully picture how microbes communicate with the brain is not yet fully elucidated and further investigations are undertaken to address unresolved questions, which will help for the development of

	IV. Materials and methods
	IV.A. Generalities
	IV.A.1. Reagents and chemicals
	All commercial reagents were purchased from Carlo Erba Reactifs and /or from
	Merck_Sigma Aldrich (Val de Reuil, France and St Quentin Fallavier, France). 5-CQA was
	acquired from Acros Organics (Italia) (CAS number 327-97-9) and trans-CA, esculetin, were
	acquired from Merck_Sigma Aldrich. BHI (Brain Heart Infusion) broth was purchased from
	Bio-Rad (France). Ethyl acetate (reagent grade) and acetonitrile (≥ 99.9%, LC-MS grade) was
	obtained from Merck_Sigma Aldrich. Ultrapure water was obtained using an EasyPure
	(Barnstead, USA) water purification system. Phosphate buffer solution (PBS) was prepared at
	0.1 M by mixing 95% of 1.36% w/v KH2PO4 and 5% of 1.78% w/v Na2HPO4.2H2O and the
	pH was adjusted to 5.8. diethylacetic acid (= 2-ethylbutyric acid) (Sigma).
	Chapter IV
	, humans may respond differently to
	polyphenols ingestion, due to the high inter-individual variability in their gut microbiota profile,
	which supports the development of personalized treatment strategies based on an individual's
	metotype (Blasco et al., 2021).
	Nevertheless, larger intervention studies should be carried out, to move from proposals
	of personalized polyphenol-based dietary strategies to global ones. Recently, the combination
	of a prebiotic, e.g. polyphenols, and a suitable probiotic, has been proposed as new tool giving

of brain disorders or whether it is a consequence (Yinwei Chen et al., 2021). Furthermore, interventions to modify the microbiota should be considered specifically for each disease. To date, the case of altered profiles of Akkermansia spp. is very intriguing. Low gut levels of Akkermansia muciniphila have been detected in metabolic diseases, including diabetes and obesity (Cani, 2018), whereas MS is characterized by an overabundance of this bacterium

[START_REF] Kasper | Exploring the Role of the Microbiome in Multiple Sclerosis[END_REF]

. Therefore, it can be assumed that there is a range of balance in the presence of this bacterium which is link to gut health. Indeed, this type of microorganism that is present in small amounts in the gut microbiota could be the key to understanding the link between gut bacteria and host health and disease. Studies should consider the bacteria-bacteria complex, for successful polyphenol intervention, as bacteria do not function individually in the gut but in a complex ecosystem (Khan et al., 2021). It is not yet fully established what is a 'healthy gut microbiota`, but scientists consider a stable and diverse of microbiota to be present in the gut (rise to the concept of synbiotics (Swanson et al., 2020; Westfall and Pasinetti, 2019). In this context, our findings could be of great interest for the development of novel synbiotics related to L. reuteri and 5-CQA, in favor of esculetin production. intervention strategy based on the gut microbiota (Chakrabarti et al., 2022). Thus, the polyphenols-gut-microbiota brain axis may be an intriguing pathway by which phenolic compounds, as natural products, modify brain activity and thus alleviate brain disorders, going beyond the classical antioxidant activity. IV.A.2. Bacterial strains The bacterial strains used in our study were Lactobacillus reuteri DSM 20016, Bacteroides fragilis ATCC 25285 and Bifidobacterium longum subsp infantis DSM 20088.

  addition, we do co-culture L reuteri with B. fragilis at approximately equimolar concentration, based on OD 600 and CFU results. The first contact of bacteria with polyphenols

	min, 13% B, linear; 21-27 min, 13% B isocratic; 27-39 min, 38% B, linear; 39-47 min, 50%
	B, linear; 47-58 min, 90% B, linear; 58-61 min, 90% B; followed by washing and
	reconditioning the column. UV-visible detection covered the 240-600 nm wavelength range.
	The whole effluent from the UV-Visible detector was injected in an ESI (Electrospray
	Ionization) source. The source parameters were negative ion mode with the following
	parameters: spray voltage (4.2 kV), capillary voltage (-41 V), sheath gas (66 arbitrary units),
	auxiliary gas (10 arbitrary units), and capillary temperature (250 •C). The nebulizing gas was
	nitrogen, and the damping gas was helium. MS spectra were acquired in full scan, negative
	ionization mode in the m/z 50-2000 range. For the MS 2 experiment, collision energy of 35 eV
	was used. Data obtained were processed with the Xcalibur 1.2 software (Finnigan Corp., San
	Jose, CA, USA).
	refered as T0. The biotransformation study by co-culture was carried out in the same condition
	as describe above.

IV.C.5. Mzmine and GNPS data treatment Data obtained from low resolution mass spectrometry were first processed with MZmine 2.53. The Agilent files were converted into mzXML format with the software MSconvert, part of the ProteoWizard package. After uploading into the MZmine workflow,

, 10 -6 and 10 -7 are inoculated in duplicate by spreading on agars. The dishes were incubated

Chapter I

Chapter II reuteri DSM 20016 (Figure 36). The final step of ring closure can occur putatively spontaneously followed by classical re-aromatisation to afford esculetin. Moreover, we have observed the presence of hydroxyacetone [START_REF]Classification, Physicochemical Principles, Mechanisms, and Electron Transfer (ET)-Based Assays[END_REF]. The last metabolite detected was butyrolactone (5) butyrolactone or GBL). Compared to the standard conditions (Figure 45), B. fragilis produced the same metabolites during 30 h of anaerobic incubation, despite the absence of isocaproic acid (8). In the same manner, we observed an important decrease in the production of hydroxyacetone by B. fragilis under all conditions (data not shown). On the other hand, the production of isovaleric acid showed an increasing trend versus time Figure 46. Interestingly, we could note,

)

in the control sample at T6, the detection of a new metabolite γ-aminobutyric acid (9) (GABL) (= gamma-aminobutyric lactam) highlighting the evolution in the time of the production profile of B. fragilis independently to the 5-CQA supplementation Indeed, GABL has been highly produced in T24h, both in presence of 5-CQA and in the control.

However, the highest production of this metabolite was at T30, and its peak area reached the values of 1.3x10 8 uAU, compared to T6 and T24, with peak area of 9. The bacterial extracts were injected with a concentration of 2 mg/mL while enzymatic and electrochemical solutions were directly analysed without treatment. The volume injection is of 2 µL. The solvent system was a gradient of A (0.1% formic acid in ultrapure water) and B (0.1% formic acid in acetonitrile), which were filtered on a 0.45 µm GH Polypro membrane (VWR, Fontenay sous Bois, France), respectively, before being used. An elution gradient was applied as follows with a flow rate of 0.2 mL/min: initial, 3% B; 0-3 min, 7% B, linear; 3-21 cells [START_REF] Vernay | Bacteroides fragilis prevents Salmonella Heidelberg translocation in co-culture model mimicking intestinal epithelium[END_REF]. Caco-2/HT29-MTX/ endocrine differentiated NCI-H716 cells and RajiB co-culture were maintained for 7 days in DMEM.

IV.E.3. Evaluation of the activity of esculetin, 5-CQA and CA on the quadricellular model Stock solution (2 mM) of 5-CQA, CA were prepared in PBS, while esculetin was firstly diluted in DMSO then in PBS in order to obtain a final concentration of DMSO as 0.12% Subsequent dilutions were done to obtain two solutions at 300 µM and 15 µM. Then, 50µL of the corresponding concentrations (2 mM, 300 µM, 15µM) were diluted in culture medium (250µL DMEM) in order to obtain each compound at the following concentrations: 1mM, 50 µM and 0.1 µM. These solutions were added to the apical side of the culture system and incubated for 24 h. Simultaneously, 50 mL of PBS and 50 mL of (DMSO+PBS) were added in cells as controls. After 24 h both, apical and basolateral contents were collected, and then 350 µL of lysis buffer were added to the cells for RNA harvesting. All samples were stored at -80°C for further analysis.

IV.E.