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Un système de régulation de type quorum sensing de [i]Streptococcus thermophilus[/i] actif en milieu solide et en matrice laitière. 22ème édition du colloque du Club des Bactéries Lactiques

Contrairement aux idées reçues qui résument les propriétés nutritionnelles d’un aliment à sa seulecomposition, de nombreux travaux récents ont démontré que la structure de la matrice alimentaire est l'undes facteurs clés qui contrôlent la digestion des aliments et, par voie de conséquence, la cinétique de miseà disposition des nutriments. Un aperçu de différentes études in vitro ou in vivo menées sur une grandevariété de produits laitiers sera présenté pour illustrer ce fait. Par exemple, la comparaison des cinétiquesde digestion de produits laitiers modèles (laits crus, pasteurisés, yaourt et fromage) de compositionidentique mais de structure différente montre une forte influence de l'état physique du produit sur lavidange gastrique qui affecte fortement la cinétique d'hydrolyse des protéines laitières et labiodisponibilité des acides aminés. De façon similaire, la digestion par le nouveau-né prématuré du laithumain soumis à un procédé d'homogénéisation indique clairement une augmentation de la lipolysegastrique par rapport au même lait non transformé. L'agrégation des protéines due aux effets cumulatifsde plusieurs traitements thermiques pendant la fabrication des préparations pour nourrissons a tendance àaugmenter la résistance des caséines à la digestion. Toutefois, bien qu'il soit maintenant établi que lastructure des produits laitiers contrôle la cinétique d’hydrolyse des protéines et lipides, d'autres travauxsont nécessaires pour déterminer les conséquences physiologiques générées par les différences decinétique observées.

The food matrix structure is one of the key drivers to control the fate of food in the digestive tract. The behaviour in the gastrointestinal tract of milk protein matrices of identical composition but different micro and macrostructures was investigated in vivo. Six multi-canulated and catheterized mini-pigs were fed 6 different dairy matrices (raw and heat-treated milks, acid and rennet gels). Effluents from the duodenum and mid-jejunum as well as plasma from the abdominal aorta were collected over 7h. Structure was shown to drive the time of residence of food in the stomach. Liquid matrices generated a fast and intense peak of proteins in the intestine and amino acids in the bloodstream whereas the behaviour of rennet gels was opposite. Controlling the time of residence of food in the stomach by playing on its structure allows to design products with a fast/slow release of nutrients.

Within the dairy matrix, milk fat globules play a crucial role. How do they interact with other components of the matrix and what opportunities do they offer the dairy industry in terms of new product development?

Les protéines du lait constituent une véritable malle au trésor. Les avancées scientifiques actuelles sur le lait mettent de plus en plus en exergue la plasticité des protéines laitières, souvent décrites comme l’or blanc du lait. La capacité de ces protéines à former des assemblages aptes à fonctionnaliser des interfaces, eau-huile et eau-air, pour obtenir des émulsions et des mousses ; à texturer les produits ou encore à vectoriser des molécules d’intérêt offre un énorme potentiel d’innovation. Ce potentiel s’inscrit dans la tendance du Clean Label et la création de nouveaux produits laitiers 100% lait.Le projet PROFIL (PROtéines Fonctionnalisées pour l’Industrie Laitière) a été bâti sur ces extraordinaires découvertes. Il réunit sept partenaires académiques et un consortium de 10 industriels laitiers regroupés au sein du consortium BBA.L’objectif de la journée du 21 septembre est de partager avec les chercheurs, académiques et industriels les résultats marquants de PROFIL qui seront présentés par six doctorants.

International audience; Calcium is an essential mineral for the organism. Numerous calcium sources are available to enrich food but only calcium carbonate has been well studied. Calcium bioaccessibility depends mainly on solubility. Furthermore in vitro digestion allows to determine bioaccessibility and to identify the key parameters affecting it. This study focused on the influence of the gastro-intestinal pH, the food structure and the calcium source on the bioaccessibility of the nutrient. Four calcium sources (calcium carbonate, calcium citrate malate, dairy calcium phosphate and calcium bisglycinate) were added at 0.5% (w/w) in water (control), and in whey protein matrices as the liquid form or with heating to induce a gel. These matrices were digested under static conditions in duplicate, with the harmonized INFOGEST protocol [1]. Soluble and ionic calcium, and pH kinetics were followed during in vitro digestion. The kinetics were analyzed by nonparametric tests with repeated measurements. Results showed that all calcium sources were soluble in the matrices, mainly as ionic form (Ca2+), during the gastric phase at pH 3, but that calcium become insoluble in the intestinal compartment at pH 7. This rise in calcium insolubility led to a fall of the nutrient bioaccessibility from 20 to 80%. Dairy calcium phosphate had a distinct behavior with an important fall of solubility in the intestinal compartment due to strong ionic interactions between calcium and phosphate. In addition, the organization of whey proteins in the food matrix had no effect on solubilisation of calcium and therefore on its bioaccessibility. These results provide new insights to better understand bioavailability differences between calcium sources and matrices. Further in vivo digestion experiments on a pig model will be achieved in order to confirm bioaccessibility and to measure the bioavailability of those different sources of calcium. It will finally provide a relation between the results from in vitro and in vivo models of digestion.

Our goals :- To understand the mechanisms of breakdown of food matrices and their constituents in the gut and identify the beneficial/deleterious food components released during digestion- To determine the impact of the structure of food matrices on these mechanisms- To model these phenomena in order to develop a reverse engineering approach

Rapport Recherche et Innovations pour l'aliment et les bioproduits du département CEPIA INRA 2017; Proteins are the cornerstones of the structural and textural properties of food, whereas the production of peptides derived from these proteins is often seen as a secondary factor. The production of peptides varies according to the type of proteolytic enzymes present in the cheese, making it possible to change its texture and some of its stretching properties. Hence, we looked for the types of peptides that could be involved in the texture changes in dairy model matrices. We performed an in silico study on a “peptide” dataset previously obtained from cheeses in order to select peptides of interest, derived from caseins, the main milk proteins, and especially involved in the functional properties of cheese. Peptides with the corresponding physico-chemical characteristics were produced using two enzymes, trypsin and the endopeptidase Glu-C. They were reincorporated into casein matrices that varied in terms of their total protein concentration and the proportion of peptides within the matrices, in order to scan a range of dairy matrices, from liquid to highly viscous gels, under neutral and acidic conditions. We thus established relationships between peptides with specific characteristics (size, charge and/or hydrophobicity) able to interact with the casein matrix and to change its structural and textural properties. We showed that these properties depended on: • The type of peptides produced; • Their proportion in both the hydrolysates and the casein matrices; • The total concentration of proteins within the matrices; and • The physico-chemical conditions of the formation of the gels Finally, we validated the lasting impact of these peptides on cheese texture in semi-hard model cheeses. Our results pave the way to prepare dairy matrices with specific techno-functional properties by adapting traditional manufacturing processes or by creating new ingredients, as examples: (i) multi-functionalising matrices with peptides in order to induce changes in texture, as well as providing attractive health bioactivities; (ii) conducting in situ hydrolyses either by adding specific exogenous enzymes or by using bacteria with specific enzymatic equipment; and (iii) producing ingredients made of casein hydrolysates with modified functionalities.; Les protéines sont à la base de la structuration et des propriétés de texture des aliments, tandis que la production de peptides à partir de ces protéines est souvent considérée comme un facteur secondaire. Lors de la fabrication de fromages, il a été établi que la production de peptides varie selon le type d’enzymes protéolytiques présentes. Nous avons donc recherché quels types de peptides pouvaient être impliqués dans les changements de texture. Nous avons réalisé une étude in silico sur des jeux de données « peptides » acquis sur les fromages, afin de sélectionner des peptides d’intérêt, issus des caséines, plus particulièrement impliqués dans les propriétés fonctionnelles des fromages. Avec deux enzymes, la trypsine et l’endopeptidase Glu-C, nous avons produit des peptides ayant les mêmes caractéristiques physico-chimiques d’intérêt. Nous les avons réincorporés dans différentes matrices, de concentration totale en protéines et en proportion de peptides variables, pour balayer des gammes de matrices laitières allant de liquides à des gels très visqueux, neutres ou acides. Nous avons ainsi établi des liens entre les peptides possédant des caractéristiques spécifiques (taille, charge et/ou hydrophobie) susceptibles d’interagir avec la matrice caséinique et d’en modifier les propriétés de structuration et de texturation. Nous avons montré que ces propriétés dépendent • du type de peptides produits, • de leur proportion dans l’hydrolysat et dans les matrices • de la concentration totale en protéines des matrices laitières et • des conditions physico-chimiques de formation des gels. Enfin, nous avons validé dans des fromages modèles de type pâte pressée non cuite l’impact durable de ces peptides sur la texture. Nos résultats offrent de nouvelles pistes pour préparer des matrices laitières avec des propriétés techno-fonctionnelles spécifiques en adaptant les fabrications traditionnelles ou en créant de nouveaux ingrédients et par exemple, (i) multi-fonctionnaliser les matrices avec des peptides afin d’obtenir des modifications de texture et de nouvelles bioactivités; (ii) mettre en oeuvre in situ des hydrolyses soit par ajout d’enzymes exogènes spécifiques soit par utilisation de bactéries avec des équipements enzymatiques particuliers; ou encore (iii) produire des ingrédients à base d’hydrolysats caséiniques avec des fonctionnalités modifiées.

This thesis was realized to respond to two particular objectives a) to determine the presence of bioactive peptides with activities as antioxidants and inhibitors of the angiotensin converting enzyme (ACE) in Mexican cheeses; and b), to observe the effect of composition and structure of dairy matrices in survival of dairy microorganism through digestion. Two Mexican cheeses were analyzed separately: Cotija cheeses ripened 6 months; and Fresh goat cheese (unripened) produced from raw and pasteurized milk. Studied cheeses had important antioxidant activity that was higher in fractions with smaller peptides. Young Cotija cheese had similar values than Fresh goat cheese. But activity increased throughout ripening (because of the production of soluble peptides). Milk pasteurization (Fresh goat cheeses) did not affect the antioxidant activity. This activity was correlated with the hydrophilic peptides. Also, both cheeses possess ACE inhibitor activity that was higher in Fresh goat cheese than in young Cotija cheese. This inhibitor activity was more correlated with the hydrophobic peptides. Pasteurization of Fresh goat cheeses increased the production of hydrophobic peptides. The release of bioactive peptides in cheeses has always been considered as dependent of the proteolysis caused by cheese microbiota. Some of these microorganisms are capable to survive digestion. It has been hypothesized that the presence of a food matrix could enhance their resistance through digestion.We measured the viability of three dairy microorganisms; Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during (in vitro and in vivo) digestion. Microorganisms were included in liquid and gel dairy matrices with and without fat: Skim milk (SM), whole milk (WM), rennet gel of skim milk (GSM) and rennet gel of whole milk (GWM). In vitro digestion was carried on with a dynamic digester (DIDGI) that simulated the stomach, duodenum and small intestine. Parameters of digestion were the same for all the matrices tested. The degradation of the dairy matrices through in vitro digestion was analyzed by electrophoreses and GC-Mass. Confocal laser scanning microscopy (CLSM) was performed to observe matrix microstructure.During in vitro digestion, the microorganisms had different survival rates; S. thermophilus was highly sensitive to gastric stress, and was not found in the duodenal compartment. B. auranticum was moderately sensitive to gastric stress but resistant to duodenal stress. H. alvei was highly resistant to both stresses. Despite its buffer capacity, we did not observe any effect of the dairy matrices on microorganisms survival. CLSM images, probed the effect of low pH on microorganisms survival. However our in vivo analyses failed to confirm in vitro observations and tested microorganisms were not detected.; Cette thèse eu deux objectifs: A) Déterminer la présence de peptides bioactifs avec des activités antioxydantes ou inhibitrices d´enzyme de conversion de l'angiotensine (ECA) dans les fromages mexicains. B) Observer l'effet de la composition et de la structure de matrices laitières dans la survie des microorganismes laitières pendant la digestion. Les fromages ont été analysés séparément: fromage Cotija, affiné 6 mois et fromage de chèvre frais (non affiné) produit à partir de lait cru et pasteurisé. L´activité antioxydante a été plus élevé dans les fractions contenant des petits peptides. Le fromage Cotija jeune a donné des valeurs similaires au fromage de chèvre frais mais l'activité augmente pendant l´affinage (en raison de la production de peptides solubles). La pasteurisation n'a pas impacté l'activité antioxydante dans les fromages de chèvre frais. Cette activité a été corrélée avec les peptides hydrophiles. Les deux fromages ont une activité inhibitrice de l´ECA qui a été plus élevée dans le fromage de chèvre que dans le fromage Cotija jeune. Cette activité était corrélée avec les peptides hydrophobes. Les fromages de chèvre frais, fait avec du lait pasteurisés, ont une concentration en peptides hydrophobes plus élevé. La libération de peptides bioactifs dans le fromage a toujours été considérée comme dépendante de la protéolyse par action des microorganismes. Certains de ces microorganismes sont capables de survivre à la digestion et l'hypothèse que la présence d'une matrice alimentaire pourrait améliorer leur résistance pendant la digestion a été testée. Nous avons mesuré la viabilité des microorganismes; Streptococcus thermophilus, Brevibacterium aurantiacum et Hafnia alvei pendant la digestion (in vitro et in vivo). Les microorganismes ont été inclus dans des matrices laitières liquides et gélifiées avec et sans matière grasse: lait écrème (SM), lait entier (WM), gel présure de lait écrèmé (GSM) et gel présure de lait entier (GWM). La digestion in vitro a été réalisée sur un digesteur dynamique (DIDGI) qui simule l'estomac, le duodénum et l'intestin grêle. Les paramètres de la digestion ont été les mêmes pour toutes les matrices. La dégradation des matrices pendant digestion in vitro a été analysée par électrophorèse et GC-Mass. La microscopie confocale à balayage laser (CLSM) a été aussi réalisée pour observer la microstructure. Pendant la digestion in vitro, il a été montré que S. thermophilus était très sensible au stress gastrique, et n'a pas été trouvé dans le compartiment duodénale. B. auranticum était modérément sensible au stress gastrique, mais résistant au stress duodénale. H. alvei était très résistant aux deux stress, par contre aucun effet des matrices laitières n'a été observé. Les images de CLSM ont montré l'effet de l´acidité sur la survie des microorganismes. Mais les analyse in vivo n’ont pas réussi à confirmer les observations in vitro parce que les microorganismes testés ne sont pas détectés.

Food products are complex matrices. They contain molecules from several families that interact with each other to generate specific structures and functionalities. These molecules may include proteins and peptides resulting from protein hydrolysis, e.g. in cheese. It is still difficult to establish a clear relationship between a pool of peptides and their techno-functionalities such as emulsifying, foaming, gelling or even texture properties within a complex matrix. Among the data usually known, there is the degree of hydrolysis that provides information about the average size of the peptides formed. A high degree of hydrolysis induces the production of a pool of small-size peptides (less than 20 amino acids). Advances in identification methods have made the identification of peptides present in food products possible. The size, charge, hydropathy index and isoelectric point of the peptide, as well as its secondary structure, can be calculated on the basis of the peptide sequence. The aim of this study was to determine which physicochemical and structural characteristics of peptides are involved in the cheese stretchability. This work is a re-evaluation, using multivariate exploratory analysis, of the experimental data obtained from a former study (Sadat-Mekmene et al., 2013) in which one typical functionality criterion of Swiss-type cheese (stretchability) was measured and peptides were identified. This methodology, based on Principal Component Analysis and Correspondence Analysis, is one way to establish a relationship between peptide characteristics and their techno-functional property within a complex dairy matrix. This statistical approach showed that the peptides predominantly involved in cheese stretchability were a mixture of both hydrophobic and hydrophilic peptides and that they are large enough to interact with each other and with native proteins. This approach could be applied to better understand the impact of peptides on various food matrices and food techno-functionalities.

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix

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Collection : Synthèses; Lipid digestion takes place in the stomach (gastric phase) then in the small intestine, proximal, or duodenal (intestinal phase). Lipolysis is catalyzed by different lipases at the lipid/water interface, and its smooth functioning will guarantee optimal absorption of different lipid nutrients. The properties of this interface (quantity, composition, organization), affecting the activity of lipases, vary considerably within the milk and dairy products according to the technological processes used (mechanical treatments such as homogenization, heat treatment). The organization of lipids in dairy matrices and the composition of their interface can therefore affect their hydrolysis by digestive enzymes and their metabolism by the body. After describing the lipids of the different dairy matrices, we will discuss about their digestion, and then their metabolic fate highlighting key points about the relationship between their structure and health.

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