Thèse la Lutéine Issue de Microalgues sur des Cellules Humaines de la Production à l'Évaluation In Vitro H/F - Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Sciences de l'ingénierie et des systèmes École doctorale : Interfaces : matériaux, systèmes, usages Laboratoire de recherche : LGPM - Laboratoire de Génie des Procédés et Matériaux Direction de la thèse : Victor POZZOBON ORCID 0000000315300834 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-22T23:59:59 La lutéine est un caroténoïde présent chez les organismes photosynthétiques qui possède des effets bénéfiques importants sur la santé, en particulier pour les yeux et le cerveau. Cependant, la consommation de légumes chez l'humain est trop faible pour couvrir les besoins en lutéine. Il faut donc développer des stratégies afin d'augmenter l'apport en lutéine. Dans ce contexte, les microalgues apparaissent comme une solution prometteuse (contenant 100 à 1000 fois plus de lutéine que les légumes et pouvant être conditionnées de manière similaire à la levure de bière), tout en nécessitant peu de ressources et en offrant des bénéfices environnementaux tels que la capture du CO. Néanmoins, bien que notre laboratoire ait obtenu des résultats solides dans la production de microalgues riches en lutéine, le chemin est encore long avant de pouvoir proposer un produit fini. Parmi les actions nécessaires, l'étude de l'absorption de la lutéine au sein du tractus gastro-intestinal humain est primordiale.Le projet vise à répondre à ce besoin de recherche en produisant des microalgues riches en lutéine et en évaluant leurs effets biologiques sur des lignées cellulaires humaines. Il débutera par la culture de microalgues, suivie d'expériences in vitro sur des cellules intestinales humaines et des cellules de tissus cibles afin d'évaluer leurs effets antioxydants, anti-inflammatoires et protecteurs face au stress oxydatif. Notre recherche s'intéressera également à la biodisponibilité de la lutéine à l'aide de modèles de digestion simulée, afin de déterminer la fraction effectivement absorbée et ses interactions avec d'autres composés des microalgues tels que les lipides et les vitamines. Enfin, l'étude examinera les effets combinés de la lutéine au sein d'une matrice microalgale réaliste afin d'identifier d'éventuelles interactions synergiques ou antagonistes susceptibles d'influencer son efficacité.

Dans l'ensemble, ce travail à pour but de générer de nouvelles connaissances sur le comportement de la lutéine issue des microalgues dans les systèmes biologiques et à évaluer la pertinence des microalgues en tant qu'aliments servant de source de lutéine. Les résultats attendus sont : une meilleure compréhension de la biodisponibilité de la lutéine, l'identification des niveaux d'apport efficaces, ainsi que des indications sur les combinaisons alimentaires optimales. Au-delà de ses contributions scientifiques, ce projet soutient également le développement de systèmes alimentaires durables et d'ingrédients fonctionnels, positionnant les microalgues comme une alternative viable aux sources conventionnelles de lutéine, tout en contribuant à la durabilité environnementale et économique. Since the Second World War, the human population has dramatically increased. In spite of technological progress, this growth and the associated quality of life improvement have put great stress on our ecosystem (Díaz et al., 2019). Pressures on resources such as fossil fuels, water, arable lands, and biodiversity are the most striking examples. A global indicator of this trend is the overshoot day (Fig. 1 - see pdf), which decreases every year.

In addition to pressuring our environment, the modern lifestyle (especially sedentary and excessive consumption of transformed products) is associated with negative health consequences. The main ones are cardiovascular diseases, diabetes, and, one of the most common is metabolic syndrome (Eckel et al., 2005).

Microalgae are now considered a relevant tool to tackle most of these challenges. They are a promising high quality source of food and feed (proteins, micronutrients) as well as bio-sourced molecules with numerous applications in the cosmetics and pharmaceutical sectors (pigments, texturizers, antioxidants) (Levasseur et al., 2020). Besides, their cultivation comes with environmental benefits: carbon dioxide and NOx sequestration, water pollution remediation (nitrate, nitrate, and phosphate), and possible valorization as biofuels of extraction processes' leftovers. Finally, they can be cultivated on non-arable lands, avoiding competition with current food-producing cultures.

Focusing on human health, the pigments produced by microalgae are of great interest. They hold numerous beneficial properties ranging from proven antioxidant effects to alleged anti-cancer properties. Astaxanthin is their flagship. It is known to be the most potent natural antioxidant, 7000 times more efficient than beta-carotene. Still, it is notoriously hard to produce and extract. Furthermore, the associated market is unclear. This hype casts a shadow onto lutein (Fig. 2 - see pdf), another carotenoid pigment with observed health benefits, a growing market, and an acknowledged need for production process improvement (Camarena-Bernard & Pozzobon, 2024).

Lutein is one of the very rare molecules capable of crossing the blood-brain barrier, which explains how it can access and positively affect the eye. For a long time, scientists were unsure of additional systemic benefits associated with lutein. Recent studies (observational, and meta-analyses) have ascertained them (Buscemi et al., 2018; Stringham et al., 2019). Lutein intake is associated with improved cognitive performances in all stages of life (from infant to elderly). The hypothesized mechanism is the following: plasma lutein crosses the blood-brain barrier and accumulates in the brain, where it acts as an antioxidant and prevents neuron damage. Consequently, neuronal efficiency increases, which translates into better memory, higher verbal fluency, and slowing down of Alzheimer's disease.

Unfortunately, the Western diet contains only 1.7 mg of lutein per day, while 6 to 14 mg/day is recommended (Hajizadeh-Sharafabad et al., 2019). There is, therefore, a need for a specific lutein production delivering food supplements. Currently, lutein is extracted from marigold flowers. This process is regarded as suboptimal in various ways: it requires arable lands and large quantities of water, the production is seasonal, the associated work is tedious, and the produced lutein is esterified, which lowers its bioavailability. Furthermore, the final product of lutein extraction (a pill) only addresses a niche market (as opposed to being available for the general population). Our group has been promoting food-grade, EFSA- and US FDA-approved microalgae as alternative lutein producers for half a decade. Today, we aim at going one step further and begin the exploration of the effects of microalgal lutein on humans.
This Ph.D. project is composed of three axis (production, baseline testing, and simulated diet testing). Together they aim at contributing to create new knowledge on the effect of microalgal lutein on human cells. The ultimate goal is to advise on the relevance of the inclusion on microalgae in a diet (as a lutein vector). The following methodology will be used:

1. Producing and purifying lutein using a microalgal biotechnology process and conventional extraction and purification techniques. The objective is to obtain high purity lutein to be used for baseline experiments in the rest of the PhD.

2. Culture human cell lines (human intestinal epithelial cells for absorption tests, and another cell line as model of target tissue - specific lines to be determined by the literature survey). Once cultivated, we will expose them to stressor (e.g., oxygen peroxide) in presence and absence of the lutein. The objective is to obtain the baseline antioxidant, and anti-inflammatorys effect of lutein. The methodology will be similar to the one of Zhang et al., who investigated Naringenin (NA) protective effect (Fig. 3 - see pdf) (Zhang et al., 2022). With this data, we will be able to assess the minimal level for lutein to have an effect as well as the level above which the bioactivity gains become marginal. The human intestinal epithelial cells will be used not only to assess lutein transport rate but also potentially positive effect on the integrity of the intestinal barrier. These two parameters are important as lutein is envisioned to be delivered through the inclusion of microalgae in the diet in our proposal.

3. In vitro digestibility tests will be carried out with the lutein-rich microalgae. The objective is twofold. First, determine which fraction of the lutein that is absorbed (to be correlated with a diet intake and the two levels identified at step 2). Second, it will be used to assess the couplings with other molecules contained in microalgae and known to have a positive effect (omega-3, omega-6, ascorbate, ...). This last piece of information will help us in building a preliminary list of molecules than may have an additive, synergistic, or antagonistic effect with lutein.

4. Based on the results of the previous action, different extracts (in different solvents) from lutein-rich microalgae will be produced. The aim is to have one (or a combination) resembling the composition of the results for the in vitro digestibility assays. This most representative extract will be delivered to the cells and its effect will be assessed. Here we expect three possible outcomes (with lutein-normalized dosages for comparison):
Synergistic effect we will work to identify with which molecules lutein works in synergy by exposing cells to pure lutein + candidate molecule couples. The ultimate goal would be to advise on dietary patterns fostering lutein absorption/effect.
Antagonistic effect similarly to the synergistic effect, we will work on identify the antagonist molecule. The aim would be to advise on dietary patterns to avoid if one intends to make the most of the lutein.
No combined effect while not the most enticing outcome from a scientific perspective, it will facilitate the dose-response mechanisms analysis.
The thesis will be articulated around different axes. They could be carried out in parallel. For example, getting familiar with the experimental device could be done at the same time as the bibliography on the most relevant cell lines and associated procedures.

Literature survey
The objective of this study is to bring the student up to date on the following areas:
- microalgae cultivation (photoautotrophy, heterotrophy, mixotrophy),
- extraction procedures to recover lutein (solvent, protocols, ...),
- in vitro digestibility techniques (INFOGEST and associated quantification),
- human cell line testing (exposure to molecule, effect analysis, ;...).
The main outcome will be: the choice of the lutein-rich microalgae production process; the associated lutein recovery processes; the choice of the most relevant human cell lines; the associated test procedures; the most suited in vitro digestion process.

Microalgae production
This task has two goals. First, it will deliver the biological material used to extract and purify lutein (for baseline effect qualification), to power the in vitro digestibility (to identify what is bioavailable), and the extract formulation (to simulate the bioavailable fraction). Second, it will allow the student to become familiar with the experimental set-up, the culture protocols, and the laboratory itself (interactions with other team members, training on analytical equipment, etc.).

First, this task will require to maintain a subculture of microalgae. This seed will then be cultured in a bioreactor (5 liter) in axenic conditions. Batch or fed-batch strategies could be used depending on the ease of conduct of the culture. Afterward, the culture will be harvested, washed, and used as raw material for extraction. Then, the extraction process will be carried out. Given the large quantity of cells to process, a high-capacity ultrasonicator will be used to rupture the cells before leading a solvent extraction. Finally, lutein will be purified using Khachik's food-grade protocol (Khachik, 1995).

Test on human cell lines with purified lutein
Two types of tests can be envisioned. The first is a transport test by human colon epithelial cells (e.g., Caco-2), the aim would be to evaluate how easily lutein would enter the blood stream. The second is a test of lutein activity (as a protector). This test could also be led on human colon epithelial cells or on a second cell line (with a higher representativity of the targeted tissues). The choice of the procedure will be led based on the literature survey findings.

From a technical point of the view, this action will require human cell line culturing, differentiating, exposition to a controlled stress, exposition to the stress in presence of lutein, and deployment of analytical procedures allowing to quality the cell responses in the different conditions. While the specific details will be ascertained by the literature survey, a tentative methodology can be proposed:
1. Cell Culture and Preparation Culture Caco-2 cells under standard conditions and monitor monolayer formation and morphology to ensure healthy, confluent cells for experiments,
2. Cytotoxicity and Oxidative Stress Model Determine safe and effective lutein concentrations using the MTT assay. Establish oxidative damage by exposing cells to HO at a concentration that reduces viability by ~40 %),
3. Protective and Anti-inflammatory Evaluation Pre-treat cells with lutein and expose to HO to assess protection against oxidative damage. Evaluate lutein's potential anti-inflammatory effects and compare efficacy across concentration.

In vitro bioavailable fraction evaluation and analysis
Lutein bioavailability will be carried out using in vitro testing. Our first guess would be to use INFOGEST methodology. It will require to quantify the initial content of lutein in the microalgae (classical procedure available in our laboratory). Then the digestibility test will be performed. It will produce a liquid fraction (bioavailable for human colon epithelial cells to transport) and a solid fraction (simulated feces). Lutein quantification will be led on the two outputs in order to assess for a mass balance (intake = quantity in solid + quantity in liquid). If the balance does not comes near a 100 %, it would suggest degradation. We would then carry out intermediary sampling to identify where lutein is degraded. Ultimately, the composition of the bioavailabe (liquid) fraction will be qualified (HPLC and Gas Chromatography analyses).
This action will also produce the material (digested fraction) used in the next one (evaluating the transport by epithelial cells and the effect on the targeted cell line).

Transport evaluation
Human colon epithelial cells will be put in contact of the bioavailable fraction obtained from microalgae in vitro digestion. Lutein transport rate will be examined and compared to the one obtained with pure lutein. The objective is to assess for possible synergistic or antagonistic effects on the transport. Also, we will be able to analyze what kind of molecules are transported in parallel of lutein (HPLC and Gas Chromatography analyses).

Evaluating the simulated matrix effect
Once in vitro digested lutein transport rate has been measured, the effect of the bioavailable fraction will be analyzed. We will carry the tests (antioxidant, anti-inflammatory activity measurements) on epithelial cells (the one transporting the lutein to the blood serum) and the targeted cell line.

If the interpretation of the effects are unclear (which molecules act in synergy, or as antagonists to lutein). We will use the extracts (mentioned above) to isolate their contribution and create simpler analogs (e.g., mixture of two extracts to analyze lutein & lipid fraction, but without aqueous fraction).

During the thesis
Frequent reporting, in the form of written reports and oral presentations, will be expected from the doctoral student. This will enable her/him to accumulate material already written and thus ease the writing of her manuscript. At least one publication in an international peer-reviewed journal will be a prerequisite for the defense of the thesis work. Three articles seem within reach.

Le/la candidate doit posséder des compétences dans les domaines suivants : culture de lignées cellulaires humaines, essais in vitro et analyses associées. Sur le plan personnel, nous recherchons une personne dynamique, curieuse, autonome et capable de travailler en équipe. De manière générale, nous accordons une grande importance aux qualités personnelles ; les compétences techniques seront acquises au cours du projet.