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International audience; Swiss cheeses have been produced for centuries in a traditional way and their typical opening indicates the presence of propionibacteria. These last are indeed present in raw milk, their growth is favored by the specific technology of Swiss-type cheeses, and their high population in the ripened cheese is responsible for its characteristic sensory properties, including the opening and the aroma. In addition to this long history of safe use and consumption, they constitute a source of nutraceuticals. The specific propionic fermentation leads to the production of short chain fatty acids as main final obligatory products. These exert beneficial effects including nourishment of digestive epithelial cells, as well as promotion of differentiation and of apoptotic depletion of colon cancer cells. Propionibacteria also produce vitamins of the B group, which enhances the nutritional value of the food products they ferment. Some selected strains were moreover shown to produce immunomodulins, proteins able to modify the secretion of cytokines in human immune cells. These last were identified as proteinaceous components of propionibacteria surface layer. They also produce extracellular vesicles which modulate in vitro inflammation and the NFκB pathway in human intestinal epithelial cells. Accordingly, such strains modulate the immune response and mitigate inflammation. In mice, they prevent both mucositis and colitis, via such immunomodulins limiting acute inflammatory response and protecting the gut epithelial barrier. In pigs, their consumption also modulates the inflammatory response and the lymphocyte populations. Finally, clinical studies dealing with consumption of propionibacteria whey cultures suggest a beneficial role in the context of IBD. Propionibacteria strains were recently identified in the gut microbiota of healthy newborns, yet not in that of children with necrotizing enterocolitis. Indeed, propionibacteria were detected in human fecal samples, which means that propionibacteria may be traditional cheese starters or next generation probiotics. The growing knowledge of these peculiar actinobacteria, including their great strain-diversity, opens new avenues for the development of functional foods and of food supplements, in a general context of increasing incidence of ailments that include an inflammatory component. Moreover, their ability to ferment both dairy and vegetable matrices is presently investigated and the first promising advances open new perspectives for the development of functional fermented vegetable products.

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

Introduction Intestinal epithelial barrier (IEB) integrity plays a central role in the pathogenesis of inflammatory bowel disease (IBD). There is a growing interest in dietary compounds that can strengthen IEB. First, candidate strains of dairy propionic bacteria such as Propionibacterium freudenreichii are able to reduce inflammation and improve barrier strength. Knowing that certain products of omega-6 polyunsaturated fatty acid (n-6 PUFA) metabolism also reinforce IEB, and that bacteria can modulate the bioavailability of host lipid compounds, this project aims to identify strains of P. freudenreichii that could protect against colitis by strengthening IEB and increasing the bioavailability of certain n-6 PUFA. MethodsA screening of thirty-three strains of P. freudenreichii was performed based on the following criteria: 1) characterization of the ability to induce production of anti-inflammatory cytokines (IL-10) by stimulation of human PBMCs 2) identification of surface proteins (S-layer protein) responsible for anti-inflammatory properties 3) measurement of their ability to increase the bioavailability of certain n-6 PUFAs, such as conjugated linoleic acids (CLAs). The ability of the selected fermented products to prevent the inflammation-induced increase in permeability of an IEB was then studied. ResultsAll strains are able to induce IL-10 production and release CLAs. Ten strains show an anti-inflammatory S-layer profile. Some fermented products significantly prevent the inflammation-induced increase in permeability, and may involve surface proteins and bioactive lipids. DiscussionThese results will allow the selection of strains with anti-inflammatory as well as pro-repair potential to be tested in vivo to reduce colitis, with or without n-6 PUFA supplementation.

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

The role of Propionibacterium freudenreichii in mitigating inflammation has been a subject of study for many years. Lately, it has been shown that immunomodulation properties are strain- specific and that the major responsible for inflammatory modulation ofimmunomodulation by strain CIRM-BIA129 is the presence of the surface layer protein B (SlpB), which has presented immunomodulation even when expressed in other bacteria. Extracellular vesicles (EVs) are nanosized spherical structures, produced by organisms of all kingdoms, including bacteria. They have been associated with inter-organism communication, pathogenesis, competition, and immunomodulation. Recent studies were aiming to address the role of EVs, in the probiotic effects of bacteria. The properties of P. freudenreichii CIRM-BIA129- derived EVs have been investigated. EVs produced by CIRM-BIA129 cultured in milk ultrafiltrate medium (UF) have been characterized by regarding size and morphology. UF-derived EVs displayed a monodisperse pattern with a modal size of 84.80 ± 2.34 nm. They are composed of a wide variety of proteins, mainly involved in metabolic processes, cellular processes and signaling, and storage and processing of information. A, among these proteins, SlpB was found in high abundance. P. freudenreichii EVs were able to inhibit, in a dose dependent manner, the increase of IL-8 production in HT-29 cells induced with LPS, due to NF-KB pathway inhibition, without causing cell cytotoxicity. EVs produced by a CIRM-BIA129 mutant strain with a knockout for SlpB showed a less efficient reduction in IL-8 production. Results have shown that the environmental conditions are able to modify EVs content and, consequently, their immunomodulatory effects. A change in the growth medium, from UF to YEL (yeast extract-lactate) showed a lower production of EVs, with a slightly larger size. EVs produced in YEL did not perform as well in the inhibition of the NF-KB pathway and had no effect on IL-8 production. Recent results have shown that P. freudenreichii EVs were able to protect Caco-2 cells from inflammation-induced excessive increasing permeability. These Altogether, these results show that Evs produced by beneficial propionibacteria are able to generate immunomodulationtrigger immunomodulation, similar to the parental strain. As for the SlpB role in EVs immunomodulation

International audience; IntroductionGut 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 Dairy propionibacteria strains were tested in vitro on human PBMCs with respect to their ability to induce immunomodulatory cytokines. Selected strains were then tested in vitro with respect to their ability to protect mice from TNBS-induced colitis, from DSS-induced colitis, and from 5FU-induced mucositis.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 (Belo et al., 2021).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).P. freudenreichii was further shown to produce extracellular vesicles (EVs), which mimic the immunomodulatory features of propionibacteria in vitro by modulating NF-κB transcription factor activity and IL-8 release in cultured human intestinal epithelial cells (Rodovalho et al., 2020). Proteomic analysis revealed presence of SlpB in these EVs.DiscussionThis 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.

In a general context of food transition, plant-based fermented products experience a considerable development. This results from evolution of consumers’ habits, demanding healthy products with a low environmental impact. This requires research and development, and thus higher scientific knowledge. Fermented soy milks constitute a favorable alternative source of live probiotics. Lactobacillus delbrueckii subsp. bulgaricus and Propionibacterium freudenreichii are extensively used in probiotic dairy products. Their adaptation to the dairy environment has been extensively studied and documented. However, little is known about their adaptation to the soy environment. We investigated suitability of soymilk as a substratefor growth and acidification by Lactobacillus delbrueckii CIRM-BIA1592 and Propionibacterium freudenreichii CIRM-BIA129. A specific focus was made on changes in term of proteome composition, cellular morphology in comparison with the cultivation in cow milk. In order to understand such adaptation, we explored their growth and survival in soy milk ultrafiltrate and compared it to cow’s milk ultrafiltrate. The two species grew in cow milk,yet not in soy milk. P. freudenreichii did grow in soy milk when co-cultured with the lactic acid bacterium L. plantarum while L. delbrueckii did not, even in co-culture with S. thermophilus. Our results revealed major differences in bacterial morphology and proteome composition. Lactobacilli upregulated stress proteins, and downregulated cell cycle and division machinery. Regarding propionibacteria, differential expression of proteins involved in amino acid metabolism, in carbohydrate metabolism, and in stress remediation was observed. These results suggest that changing the fermented substrate may significantly affect both technological and probiotic properties of conventional dairy starters. This should be considered for the development of new fermented functional foods. Screening of a large collection of lactic and propionic acid bacteria is in progress, based on their technological and probiotic properties. Bacterial stains and consortia thereof will be selected for the production of new plant-based fermented functional foods

International audience; Introduction and Aims. Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects of selected strains within the species P.freudenreichii, S.thermophilus and L.delbrueckii. We thus hypothesized that consumption of a functional cheese, fermented by selected strains of these three species, may exert a positive effect on IBD.Methods. Strains of each bacterial species were screened based on the ability to induce regulatory IL-10 in human immune PBMC cells, or to downregulate nuclear factor kappa B (NF-κB) activation in a cultured HT-29 human intestinal epithelial cell line. We then investigated the effect of monostrain experimental cheese. We further investigated the impact two-strains experimental cheese. Finally, we produced in industrial conditions an Emmental cheese using one selected strain of each of the three species, i.e. P. freudenreichii CIRM-BIA 129 in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9. Consumption of all cheeses was investigated with respect to prevention of chemically induced colitis in mice.Results. Consumption of the experimental cheeses, or of the industrial Emmental, reduced the severity of subsequent chemically induced colitis, weight loss, disease activity index and histological score, in mice. Furtherrmore, consumption of the Emmental cheese, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17).Conclusion. Assembling immunomodulatory strains of both lactic acid and propionic acid starter bacteria leads to an anti-inflammatory Emmental cheese, as revealed in vivo. This opens new perspectives for the development of functional fermented food products for personalised nutrition in the context of IBD.

International audience; Inflammatory bowel diseases (IBDs) constitute disturbances of gastrointestinal tract that cause irreversible changes in the structure and function of tissues. Ulcerative colitis (UC), the most frequent IBD in the population, is characterized by prominent inflammation of the human colon. Functional foods containing probiotic bacteria have been studied as adjuvants to the treatment or prevention of IBDs. The selected probiotic strain Lactococcus lactis NCDO 2118 (L. lactis NCDO 2118) exhibits immunomodulatory effects, with promising results in UC mouse model induced by dextran sodium sulfate (DSS). Additionally, cheese is a dairy food that presents high nutritional value, besides being a good delivery system that can be used to improve survival and enhance the therapeutic effects of probiotic bacteria in the host. Therefore, this work investigated the probiotic therapeutic effects of an experimental Minas Frescal cheese containing L. lactis NCDO 2118 in DSS-induced colitis in mice. During colitis induction, mice that consumed the probiotic cheese exhibited reduced in the severity of colitis, with attenuated weight loss, lower disease activity index, limited shortening of the colon length, and reduced histopathological score. Moreover, probiotic cheese administration increased gene expression of tight junctions’ proteins zo-1, zo-2, ocln, and cln-1 in the colon and increase IL-10 release in the spleen and lymph nodes. In this way, this work demonstrates that consumption of probiotic Minas Frescal cheese, containing L. lactis NCDO 2118, prevents the inflammatory process during DSS-induced colitis in mice, opening perspectives for the development of new probiotic functional foods for personalized nutrition in the context of IBD.

International audience; Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines and mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e., modulating NF-κB transcription factor activity and interleukin-8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications, such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here, we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and, hence, the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical, and functional properties of EVs produced from cells cultivated on laboratory yeast extract lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant and smaller in diameter, and they displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacterium interactions. IMPORTANCE Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in hostmicrobe interactions. Recently, we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

4.1 Infant formula (IF) and follow-on (FO) powders are formulated products used for early-life nutrition. Typical processing steps used in the manufacture of IF/FO powdered formulae include batch make-up, heating, homogenization, evaporation and drying. Both IF and FO powdered formulae must be manufactured to the highest safety standards, with increasing requirements for clinically proven nutritional benefits and optimal reconstitution properties, including good visual quality with no sedimentation, free oil, un-reconstituted particles or evidence of flecking. The chapter discusses the drying of IF and FO holistically, taking into account the effects of recipe, wet-processing and dryer configuration on production efficiency and powder quality. In particular, the effects of emulsion quality and viscosity prior to drying are presented as key variables, which illustrate the importance of formulation/recipe on wet-processing and subsequent drying behavior.4.2 The global lactose-free dairy products market still increasing drives the development of new lines of products by the dairy industry. In this sense, a wide variety of dairy products with lactose-free appeals are available on the market such as pasteurized and UHT milk, yogurt, cheeses, ice-cream, dulce de leche and other dairy products. However few reports describe the effect of lactose hydrolysis on the properties of dried milk powders during production and storage. Lactose hydrolyzed dried milk powder production remains a challenge for the dairy industry because of specific technological problems. Due to the presence of the monosaccharides glucose and galactose, lactose-free powders tend to suffer stickiness, caking and browning during drying and storage. This chapter deals with the consequences of lactose hydrolysis during the production and storage of dried milk powders.4.3 Camel milk has a composition and properties quite close to human milk. Camel milk’s composition is considered superior to that of bovine milk in terms of its nutritional and therapeutic value. It contains high concentrations of several bioactive compounds that have health benefits. To achieve long-term stability and usability, many dairy-based products and ingredients are dehydrated to powder form. However, such severe heat treatments eventuate in the loss of heat-labile bioactive compounds. Protecting these bioactive compounds during the production of camel milk powder is a challenge for dairy researchers and manufactures. To maintain the activity of such compounds, low-temperature drying operations such as freeze-drying are preferred, and there are many freeze-dried camel milk powder products available on the market. However, due to the limitations of freeze-drying in the production of milk powder, freeze-drying needs to be replaced with other economic drying approaches such as spray drying. However, the application of spray drying in the production of camel milk powder is still in early stages of research, and there are only a few reported studies. This chapter describes the bioactive properties of camel milk and the potential application of spray drying to produce camel milk powder. 4.4 An innovative processing scheme for the production of permeate powders is evaluated in this chapter. It includes an overconcentration step that makes it possible to replace the spray-drying step used in conventional processes. This innovative processing scheme includes: (1) overconcentration of the permeate concentrate from 60 to 80% w/w dry matter (DM) content; (2) granulation of the overconcentrate with powder up to 88% w/w DM; and (3) drying of the granules up to 97% w/w DM. Considering only water removal, the energy savings in comparison to the conventional process were estimated in the range of 10.7 to 23.5%, and even up to 32% when considering the whole production process or the drying step alone. The feasibility of the process was validated at pilot scale, and the results showed that it leads to significant savings in energy and building requirements for a quality of powder at least equivalent to a standard powder produced using conventional technologies.4.5 Due to the variety and complexity of the fat-filled concentrates to be dried, the dairy industry has been looking over the past 20 years for effective methods, including modeling, in order to predict the drying behavior of dairy products and adjust the outlet gas temperature and humidity. In this chapter, we describe a reliable method based on physicochemical and thermodynamic properties that was developed at INRAE to achieve this objective. It combines two approaches to predict the spray-drying parameters of fat-filled powder: on the one hand, a representative function of the availability of water in the concentrate, depending on its biochemical composition, is provided by a desorption method; on the other hand, the dryer key features (evaporation capacity, air flow rates, configuration) and weather conditions are considered (Schuck, 2013). It makes it possible to obtain a reliable prediction (± 5%) of the inlet air temperature for a given product formulation, and to develop continuous-improvement approaches.4.6 A novel spray-drying process for the continuous production of probiotics was proposed. Concentrated sweet whey (up to 30% w/w dry matter) was used to both culture and spray dry Propionibacterium freudenreichii ITG P20 and Lactobacillus casei BL23. This process cuts down the steps between culturing and drying (e.g. harvesting, washing, re-suspension), increases the cell population after growth and improves spray-drying productivity and probiotic viability. The mechanisms were explored from the point of view of both bacterial stress resistance and drying process conditions. The hypertonic stress led to overexpression of key stress proteins and the accumulation of intracellular compatible solutes, which contributed to the enhanced multistress tolerance acquisition in hyper-concentrated sweet whey. The presence of protein aggregates and increased concentration of magnesium salt in the matrix may also be involved. The feasibility of scaling up this process was validated at a semi-industrial scale. A multi-stage mild-conditions drying process, coupling spray drying with belt drying and fluid-bed drying, was applied to further improve the probiotic viability to approximately 100% (>109 CFU g−1). This work opens new avenues for the sustainable development of new starter and probiotic preparations with enhanced robustness.

International audience; Cheeses exist since ancient times and are still a matter of discovery, as well as a source of innovation. Originally designed to preserve milk, their culture- and tradition-driven technology led to the empirical selection of domesticated microbial consortia. Science has later deciphered, cultivated and characterized the members of such dedicated consortia. Microbial strains can now be selected based on both technological and probiotic criteria. Beyond the traditions and the expected hedonic perceptions, the keystones of the current changes in cheese technology are indeed the way cheeses are made, the way starters are selected and conserved as well as how the cheeses are destructured in the digestive tract, according to specific population. Here we describe our advances in these different research areas and the emerging trends for future. The selection of strains of lactic acid bacteria and of propionibacteria, each combining cheese-making and probiotic skills, leads to the production of functional cheeses, in which the dairy matrix constitutes a protective delivery vehicle for immunomodulatory components. This is illustrated in the context of colitis, i.e. gut inflammation.Cheeses enriched in whey proteins, fermented with selected starters, fulfil the requirements of elderly towards essential aminoacids.Uncoupling the 2 major steps of cheese making, texturing through protein concentration, and flavouring through microbial fermentation, allows quick, safe and sustainable production of “high-tech” cheeses with reduced energy consumption and reduced coproducts production.Incorporation of vegetable proteins, and of microbes able to process them, into the cheese-making process, leads to innovative cheeses with a reduced ecological impact.All these innovations open new avenues for the development of designer cheeses adapted to evolving needs of specific populations and addressing growing ethical concerns.

International audience; Introduction Propionibacterium freudenreichii, is a probiotic bacterium, with an established immunomodulatory role. Selected strains indeed induce the release of regulatory IL-10. Extractable surface proteins of the S-layer type (Slp) are involved in such modulation, but the exact mode of interaction with the host remains unknown. Mucositis is a common side effect of cytotoxic chemotherapy characterized by mucosal injury, inflammation, diarrhea, and weight loss. We hypothesized that immunomodulatory P. freudenreichii may mitigate inflammation and that SlpB may be required for such an effect.Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. We hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host.MethodsP. freudenreichii CIRM-BIA 129, previously selected for its ability to induce IL-10 release by PBMCs, was administered orally to mice, prior to colitis induction using TNBS.The slpB gene was inactivated by insertional inactivation. Both parental wild type and mutant strains were administered orally to mice, prior to induction of mucositis using 5-FU. The SlpB protein was purified and its effect on cultured human intestinal epithelial cells investigated with respect to cytokines expression.EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, prior to physicochemical and functional characterization.ResultsConsumption of P. freudenreichii CIM-BIA129 strain protected mice against TNBS-induced colitis in mice, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages.This strain prevented 5-FU-induced mucositis in mice, regulated key markers, including the expression of Claudin-1 (Cld1) and IL-17a (Il17a), as well as the levels of IL-12 and IL-1β cytokines.The slpB gene was mutated by insertional inactivation and the absence of SlpB protein in the resulting mutant was verified. The mutant strain displayed opposite immunomodulatory effect and failed to mitigate induced mucositis.In HT-29 human intestinal epithelial cells P. freudenreichii reduced expression of IL-8 and TNF-α cytokines in LPS-stimulated cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. In the same cells, purified SlpB was shown to induce expression of IL-10 in a dose-dependent manner.Extracellular vesicles (EVs), they were purified from cell-free culture supernatants of P. freudenreichii. They showed typical shapes and sizes of EVs and contained a broad range of proteins, including SlpB. EVs modulated inflammatory responses, IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells, as well as the NF-κB pathway.DiscussionThis work emphasizes the importance of SlpB in immunomodulatory P. freudenreichii. It constitutes the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. It opens perspectives for the development of functional foods and other bioactive products in order to help preventing and/or treating inflammatory diseases, and to decrease side effects of chemotherapy.

Genomic evidence has led to the division of genus Propionibacterium into Propionibacterium and Acidipropionibacterium. They are pleomorphic rods, anaerobic to aerotolerant, mesophilic, Gram-positive and of high G+C. They produce propionic acid as a major end product from many substrates. P. freudenreichii is used as ripening culture in Swiss-type cheeses, where it produces propionic and acetic acid, CO2 and aroma compounds leading to the characteristic flavor and eyes. They also produce vitamins and other compounds of interest and may be used as probiotics. Genetic tools and genome sequences are now available for both Propionibacterium and Acidipropionibacterium genera, which will improve their applications.

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Smart design of functional fermented dairy foods . STLOpendays