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International audience; ContextGut inflammation constitutes a growing health concern in developed countries. It may consist in spontaneous ailments of the gut, involving both the host immune system and microbiota, such as IBD, including ulcerative colitis and Crohn’s disease. It may be caused by a medical treatment, such as mucositis induced by cancer chemotherapy and/or radiotherapy. It coincides with a dysbiosis including a lack of anti-inflammatory bacteria. As an example, propionibacteria are lacking in the microbiota of newborns that develop necrotizing enterocolitis.Methods and resultsWe thus focused on the immunomodulatory properties of GRAS propionibacteria. Selected strains of Propionibacterium freudenreichii induced the regulatory IL-10 cytokine in human immune cells (Foligné et al., 2010, 2013), depending on surface proteins (Le Marechal et al., 2015). Mutation of the slpB gene suppressed this immunomodulatory effect and the resulting slpB mutant induced a rather proinflammatory response (Deutsch et al., 2017). Consumption of wild-type P. freudenreichii protected from colitis induced by both TNBS and by DSS. It alleviated severity of symptoms, modulated local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages (Plé et al., 2015, 2016; Rabah et al., 2020). Accordingly, consumption of Lactococcus lactis NCDO 2118 harboring pXIES-SEC:slpB and expressing the propionibacterial SlpB reduced severity of colitis, lowered weight loss, disease activity index, shortening of the colon length, and histopathological score, compared with mice treated with L. lactis NCDO 2118 wild-type strain.In the context of mucositis induced by the chemotherapy 5-FU, P. freudenreichii prevented weight loss, reduced inflammation and consequently intestinal damages. It regulated key markers, including Claudin-1 and IL-17a genes, as well as IL-12 and IL-1β cytokines levels (Cordeiro et al., 2018). Mutant strain slpB displayed opposite regulatory effect on cld1 expression and on IL-12 levels, and failed to afford protection towards 5-FU-mucositis (do Carmo et al., 2019).ConclusionThis work emphasizes the importance of SlpB in P. freudenreichii ability to reduce both mucositis and colitis inflammation. It opens perspectives for the development of probiotic products aimed at decreasing side effects of chemotherapy and at helping treatment of colitis, thanks to GRAS bacteria.ReferencesCordeiro, B. F., Oliveira, E. R., Silva, D., H, S., Savassi, B. M., Acurcio, L. B., et al. (2018). Whey Protein Isolate-Supplemented Beverage, Fermented by Lactobacillus casei BL23 and Propionibacterium freudenreichii 138, in the Prevention of Mucositis in Mice. Front. Microbiol. 9. doi:10.3389/fmicb.2018.02035.Deutsch, S.-M., Mariadassou, M., Nicolas, P., Parayre, S., Le Guellec, R., Chuat, V., et al. (2017). Identification of proteins involved in the anti-inflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study. Sci Rep 7, 46409. doi:10.1038/srep46409.do Carmo, F. L. R., Rabah, H., Cordeiro, B. F., da Silva, S. H., Pessoa, R. M., Fernandes, S. O. A., et al. (2019). Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy. Oncotarget 10, 7198–7219. doi:10.18632/oncotarget.27319.Foligné, B., Breton, J., Mater, D., and Jan, G. (2013). Tracking the microbiome functionality: focus on Propionibacterium species. Gut 62, 1227–1228.Foligné, B., Deutsch, S. M., Breton, J., Cousin, F. J., Dewulf, J., Samson, M., et al. (2010). Promising immunomodulatory effects of selected strains of dairy propionibacteria as evidenced in vitro and in vivo. Appl.Environ.Microbiol. 76, 8259–8264.Le Marechal, C., Peton, V., Ple, C., Vroland, C., Jardin, J., Briard-Bion, V., et al. (2015). Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties. J.Proteomics. 113C, 447–461.Plé, C., Breton, J., Richoux, R., Nurdin, M., Deutsch, S.-M., Falentin, H., et al. (2016). Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese. Mol Nutr Food Res 60, 935–948. doi:10.1002/mnfr.201500580.Plé, C., Richoux, R., Jardin, J., Nurdin, M., Briard-Bion, V., Parayre, S., et al. (2015). Single-strain starter experimental cheese reveals anti-inflammatory effect of Propionibacterium freudenreichii CIRM BIA 129 in TNBS-colitis model. Journal of Functional Foods 18, 575–585. doi:10.1016/j.jff.2015.08.015.Rabah, H., do Carmo, F. L. R., Carvalho, R. D. de O., Cordeiro, B. F., da Silva, S. H., Oliveira, E. R., et al. (2020). Beneficial Propionibacteria within a Probiotic Emmental Cheese: Impact on Dextran Sodium Sulphate-Induced Colitis in Mice. Microorganisms 8, 380. doi:10.3390/microorganisms8030380.- PF induces IL-10 in human PBMCs- Surface layer extraction reduces IL-10 induction- Extracted surface layer induces IL-10- Mutation of the SlpB gene reduces IL-10 induction- PF prevents colitis- PF prevents mucositis- PF slpB mutant does not prevent mucositis- Lactococcus lactis expressing SlpB prevents colitis

International audience; Nutritional dependencies, especially those regarding nitrogen sources, govern numerous microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary, organoleptic, and health properties of most fermented products, such positive interactions have previously been studied between yogurt bacteria. However, they have never been exploited to create artificial co-cultures of LAB that would not necessarily coexist naturally, i.e from different origins. The objective of this study was to promote LAB positive interactions, based on nitrogen dependencies in co-cultures, and to investigate how these interactions affect some functional outputs, e.g. acidification rates, carbohydrate consumption, and volatile compound production. The strategy was to exploit both proteolytic activities and amino acid auxotrophies of LAB. A chemically defined medium was thus developed to specifically allow the growth of six strains used, three proteolytic and three non-proteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was co-cultured with each one of the non-proteolytic LAB strains: L. lactis NCDO2111, Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and co-cultures. Acidification, carbohydrate consumption and volatile compound production was evaluated in direct co-cultures. Each proteolytic strain induced different types of interactions: either strongly positive, weakly positive, or no interactions, with E. faecalis CIRM-BIA2412, L. lactis NCDO2125 and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations in tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to faster acidification rates, lower pH, higher raffinose utilization and concentrations in five volatile compounds. Importance: Lactic acid bacteria (LAB) interactions are often studied in association with yeasts or propionibacteria in various fermented food products and the mechanisms underlying their interactions are being quite well characterized. Concerning interactions between LAB, they have mainly been investigated to test antagonistic interactions. Understanding how they can positively interact could be useful in multiple food-related fields: production of fermented food products with enhanced functional properties or fermentation of new food matrices. This study investigates the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and non-proteolytic strains. The results suggest that proteolytic LAB do not equally stimulate non-proteolytic LAB and that the stronger the interactions between LAB are, the more functional outputs we can expect. Thus, this study gives insight into how to create new associations of LAB strains and to guaranty their positive interactions.

La texture des aliments, leur densité contribuent à protéger les bactéries qui viendront enrichir notre flore intestinale. Démonstration avec l’emmental.

The milk fat globule membrane (MFGM) is a unique interface rich in polar lipids, cholesterol and proteins, bringing specific functions in the gastro-intestinal tract. In this book chapter, new scientific advances about the structure and mechanical properties of the MFGM are reviewed, in relation with the specific composition of this biological membrane and the physical properties of milk polar lipids that are mainly due to the high amount of sphingomyelin (SM). The MFGM is a patchwork of polar lipids with lateral phase separation of high melting temperature Tm saturated polar lipids such as milk-SM (Tm=34.3°C) leading to the formation of ordered microdomains that are higher and more rigid than the surrounding fluid matrix composed of low Tm unsaturated polar lipids in which the proteins are embeded. The microdomains are reactive to thermal kinetics and can diffuse in the plane of the MFGM. Attractive interactions between SM and cholesterol are involved in the formation of complexes in a wide range of temperatures (4 to 60°C). Structural and mechanical heterogeneities in the MFGM and in milk polar lipid assemblies have been evidenced. These recent information will contribute in a better undertanding of MFGM functions and open perspectives in the valorization of milk polar lipids and MFGM-rich ingredients for food and nutritional applications.

Dietary lipids play a major role in infant nutrition, development and health. As an alternative to breast milk, infant formulae (IF) are the manufactured products given to infants. However, many differences exist between breast milk fat globules that are naturally secreted by lactating mothers and the processed lipid droplets formed under pressure during homogenization and found in IF. The lipid composition and structure of the emulsion in IF could be improved to mimick breast milk fat globules. This book chapter i) describes breast milk fat globules covered by their biological membrane (MFGM), including their functions and digestion in the gastrointestinal tract of infants, ii) presents the technological steps involved in IF preparation and the blending of oils with other ingredients leading to the final composition and structure of processed lipid droplets, iii) highlights the health benefits for infants of adding dairy lipids (Fatty acids, TAG, MFGM rich in phospholipids, sphingolipids, cholesterol and glycoproteins) in IF, iv) explains the opportunities to produce food-grade ingredients enriched in bovine MFGM and to prepare processed lipid droplets in IF bio-inspired by breast milk fat globules. The next generation of IF will integrate the advantages provided by dairy lipids to improve the quality of IF and bring nutritional and heath benefits to infants worldwide.

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering "lipocalin" protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called "stealth" siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-γ and α-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 μM) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-γ nor α-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 μM, respectively) indicating that α-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-γ and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (K d of 5.3 nM) of enterobactin over the iron-free form (K d of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW.

Milk fat physical properties, including its crystallization and melting properties, are involved in many food applications (cream, butter, cheese, and also in bakery). Over a wide range of temperatures, milk fat is a mixture of solid crystals connected in networks and embeded in a liquid phase. These crystals evolve, as a function of temperature and temperings, in terms of amount, composition and structural characteristics. This book chapter describes the crystallization properties and polymorphism of milk fat and shows how they are affected by (1) the dispersion state, i.e. anhydrous fat versus emulsified fat dispersed in numerous droplets, (2) the cooling rates and temperings, (3) shear applied during processing, (4) the presence of minor lipid compounds (FFAs, MAGs, DAGs, phospholipids), (5) the fatty acid and triacylglycerol (TAG) compositions, including milk fat from various species (human, goat, dromedary milks). Deciphering the crystallization properties of milk fat requires investigations at different scale levels, recordings as a function of temperature and time, and the combination of physical techniques. Increasing knowledge in the area of TAG crystallization yields fascinating new insights that contribute in increasing further the value of milk fat in food applications.

International audience; Donor human milk, pasteurised for safety reasons, is the first alternative for feeding preterm infants when mothers' own milk is unavailable. Breastmilk pasteurisation impact on lipid digestion and absorption was evaluated by a static in vitro digestion model for preterm infants coupled with intestinal absorption using Caco-2/TC7 cells. Lipid absorption was quantified by digital image analysis of lipid droplets, by measurement of basolateral triglyceride concentration and by analysing the expression of major genes involved. After in vitro digestion, lipolysis extent was 13% lower in pasteurised human milk (PHM) than in raw human milk (RHM). In Caco-2/TC7 cells, the number of lipid droplets was identical for both milk types, while the mean droplet area was 17% smaller with PHM. Altogether, pasteurisation decreased the pre-lipolysis of human milk. This initial difference in free fatty acid amount was only partially buffered by the subsequent processes of in vitro digestion and cellular lipid absorption.