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Le CBL (Club des Bactéries Lactiques) est une manifestation scientifique qui réunit chercheurs, enseignants-chercheurs et industriels R&D, pour échanger sur les avancées scientifiques et techniques réalisées dans le domaine des bactéries lactiques. Les thèmes abordés sont le reflet des larges potentiels de ces bactéries et de leurs applications : fermentation alimentaire, santé humaine et animale, probiotique, environnement…; International audience; En fabrication fromagère, les bactéries lactiques (BL) et propioniques sont des actrices clés de la production de métabolites conférant les qualités nutritionnelles et organoleptiques aux fromages. Pourtant, la contribution de chaque espèce à la qualité finale du fromage n’est pas totalement élucidée. L’objectif était de déterminer quelles espèces contribuent à acidifier et produire des composés d’arôme, par quelles voies métaboliques, selon quelle temporalité et aussi d’investiguer les interactions métaboliques bactériennes contribuant au fonctionnement de l’écosystème fromager.Nous avons séquencé et annoté : Lactococcus lactis subsp. lactis biovar diacetylactis CIRM-BIA1206 (LL), Lactiplantibacillus plantarum CIRM-BIA465 (LP), Propionibacterium freudenreichii CIRM-BIA122 (PF). Nous avons reconstruit les voies métaboliques et élaboré un modèle métabolique de communauté. Un fromage à pâte pressée non cuite sans croûte a été réalisé avec ces bactéries et analysé pendant la fabrication et l’affinage (4 réplicats). Des dénombrements bactériens, des dosages de sucres, d’acides organiques et de composés d’arôme [1] ont été réalisés ainsi qu’un séquençage des ARN bactériens. L’analyse des gènes différentiellement exprimés a permis de montrer à quel moment de la fabrication étaient induits les gènes impliqués dans le catabolisme du lactose et du citrate et dans les voies de synthèse de différents composés d’arômes : acides lactique, acétique, propionique, isovalérique (arôme ‘vieux fromage’), diacétyle (arôme beurré). Le catabolisme du lactose était opéré chez LL via la voie du tagatose pendant l’acidification puis via la voie de Leloir et uniquement par cette dernière voie chez LP. La production de diacétyle était effectuée par LL dès le début de la fabrication puis plus modérément par LP. Les synthèses d’acides propionique et isovalérique ont été attribuées à PF et les métabolismes correspondants induits pendant la phase d’affinage. Enfin, les voies de fermentation mixtes ayant été induites pendant l’affinage, l’acide acétique a été vraisemblablement produit par les trois espèces à cette étape. L’implémentation du modèle métabolique communautaire dans l’outil Smetana [2] a révélé les bases moléculaires du commensalisme précédemment évoqué entre BL et PF [3–5]. Les BL produisent de l’acide lactique et potentiellement du glycérol, de la sérine et de la phénylalanine au profit de PF. Ces interactions identifiées in silico restent à valider in vitro.L’ensemble de ces résultats font de la métatranscriptomique associée aux modèles métaboliques, des outils de choix pour mieux comprendre et maîtriser les métabolismes et les interactions régissant le fonctionnement des écosystèmes fromagers.

Les interactions impliquant les bactéries lactiques (BL), responsables des propriétés sanitaires, organoleptiques et santé des produits fermentés, sont souvent étudiées en association avec des levures ou des bactéries propioniques dans divers produits alimentaires fermentés et les mécanismes de leurs interactions sont assez bien caractérisés. En ce qui concerne les interactions entre BL, elles ont principalement été étudiées pour tester des interactions antagonistes. Comprendre comment les BL peuvent interagir positivement serait utile dans de multiples domaines liés à l'alimentation, comme la production de produits fermentés aux propriétés fonctionnelles améliorées ou la fermentation de nouvelles matrices alimentaires. Les dépendances nutritionnelles, notamment celles vis-à-vis des sources d'azote, régissent de nombreuses interactions positives microbiennes. De telles interactions positives ont déjà été étudiées entre les BL du yaourt. Cependant, elles n'ont jamais été exploitées pour créer des co-cultures de BL issues de biotopes différents. L'objectif de cette étude était de favoriser les interactions positives entre BL, basées sur les dépendances aux sources d’azote dans les co-cultures pour augmenter les taux d'acidification, la consommation de sucres comme le raffinose et la production de composés volatils. La stratégie consistait à exploiter les activités protéolytiques et les auxotrophies des acides aminés des BL. Un milieu chimiquement définia été développé pour permettre spécifiquement la croissance des six souches de BL utilisées, trois protéolytiques et trois non protéolytiques. Chacune des souches protéolytiques, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125 et CIRM-BIA244, a été cultivée avec chacune des souches de BL non protéolytiques : L. lactis NCDO2111, Lactiplantibacillus plantarum CIRM-BIA465 et CIRM-BIA1524. La croissancebactérienne a été suivie à l'aide de chambres compartimentées pour comparer la croissance en mono- et co-cultures. Chaque souche protéolytique a induit différents types d'interactions 2 : fortement positives avec E. faecalis CIRM-BIA2412, faiblement positives avec L. lactis NCDO2125, ou nulles avec L. lactis CIRM-BIA244. En co-cultures directes, les interactions ont conduit à des taux d'acidification plus rapides, à un pH final plus bas, à une utilisationplus élevée du raffinose et à des concentrations plus élevées en cinq composés volatils, pouvant donner des arômes intéressants. Les fortes interactions étaient associées à des concentrations plus élevées en tryptophane, valine, phénylalanine, leucine, isoleucine et en peptides. Une analyse peptidomique a permis d’identifier les acides aminés hydrophobes et à chaîne ramifiée comme essentiels dans ces interactions. Cette étude donne un nouvel aperçu des mécanismes régissant les interactions entre BL et offre des solutions pour améliorer ou concevoir des ferments lactiques.

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.

Consumption of fumonisin-contaminated foods has a negative influence on the health of humans (carcinogen; oesophageal cancer in Eastern Cape in South Africa). Lactic acid bacteria (LAB) have emerged as a promising natural detoxification agent against mycotoxins. The aim of this study was to visualise the interaction between fumonisins (FB1 and FB2) and LAB: Lactobacillus plantarum FS2, L. delbrueckii subsp. delbrueckii CIP 57.8T and Pediococcus pentosaceus D39, isolated from traditional fermented maize-based products (ogi and mahewu) using confocal laser scanning microscopy (CLSM) and to then quantify the LAB-bound fumonisin using high performance liquid chromatography (HPLC). The objective was to obtain a physically visible and quantifiable binding interaction between fumonisins and LAB strains with the aim of utilising LAB as a possible detoxifying agent. Fumonisins were derivatised using naphthalene-2,3-dicarboxaldehyde (NDA) and then combined with non-fluorescent LAB cells (viable and non-viable). For the quantification of bound fumonisins, viable and non-viable cells were incubated in the presence of predetermined concentrations of fumonisins and the level of fumonisin in the suspension was determined. CLSM showed the derivatised green fluorescent fumonisins binding to the surface of each of the LAB cells. For viable cells, L. plantarum FS2 bound FB1 most effectively while P. pentosaceus D39 bound the least level of FB1. The highest levels of FB2 were bound by L. plantarum R 1096 and the least by L. delbrueckii CIP 57.8 T. For non-viable cells, L. plantarum FS2 was also the most effective for binding both fumonisins with P. pentosaceus D39 and L. delbrueckii CIP 57.8 T being the least effective for FB1 and FB2, respectively. To our knowledge, this is the first study to visualise the interaction between LAB and fumonisins. We demonstrate that LAB isolates from indigenous fermented maize-based beverages bind fumonisins and thus present a potential strategy for their reduction in these traditional foods.

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

International audience; This study explores the ability of lactic acid bacteria (LAB) to ferment soy juice. The ability of 276 LAB strains from 25 species to ferment the principal soy carbohydrates, sucrose, raffinose or stachyose was tested in synthetic media and a soy juice. Fermented soy juices (FSJs) were characterized for their odor. Selected FSJs were characterized by targeted metabolomics. All Streptococcus, 83% of Leuconostoc and Lactobacillus and 41% of Lactococcus strains were sucrose-positive, while only 36% of all the LAB strains tested were raffinose-positive and 6% stachyose-positive. Nearly all (97%) the sucrose-positive strains fermented soy juice, indicating that an ability to use sucrose is a good criterion to select strains for soy juice fermentation. Among the most efficient acidifying strains, 46 FSJs had an odor deemed to be acceptable. FSJ composition was dependent on both species and strains: 17/46 strains deglycosylated soy juice isoflavones, the 27 S. thermophilus strains converted a mean 4.4 ± 0.1 g/L of sucrose into 3.0 ± 0.1 g/L of lactic acid versus 5.2 ± 0.1 g/L into 2.2 ± 0.1 g/L for the 18 Lactobacillus and one Lactococcus strains. This study highlights the diversity of the metabolic profiles of LAB strains in soy juice fermentation.

International audience; Lactococcus lactis ssp. cremoris and Lactobacillus acidophilus NCFM were cultured in growth media supplemented with CaCl2 to induce intracellular accumulation of Ca2+. Both strains grown with 10 mM Ca2+ demonstrated enhanced thermal stability, with increased survival after spray drying from 49.9% to 64.9% for the coccus and from 35.5% to 43.3% for the rod. The increased intracellular Ca2+ level was confirmed with murexide assay. Adding Ca2+ in the reconstitution solution could not improve the viability of spray dried cells at extended storage stages. We hypothesized that the over-presence of intracellular Ca2+ might stimulate the activity of heat shock proteins, increasing cell survival towards heat stress. The positive effect of calcium partly explained the protective mechanism of some calcium-containing protectants. The reported approach, which improves the stress tolerance of cells, can be combined with the optimization of spray drying conditions and protectant formulation for developing a consolidated powder production scheme.

National audience; A Western diet containing 50% of plant-based protein is healthier and would help to reduce the environmental impacts of food systems to contribute to meeting worldwide protein needs. However, few consumers do like plant-based protein food, 25% of consumers do like taste of soy products. Fermentation by lactic acid bacteria (LAB) is a sustainable and an inexpensive process for the preservation or raw material that can also modify taste. In this work, we screened 278 LAB in order to select strains of interest to ferment soy juice. Our findings showed that the ability of LAB to ferment soy juice is both species- and strain-dependent. We also found that some strains can improve organoleptic properties of fermented soy juices, and the study highlights the diversity of metabolic profiles of LAB in soy juice fermentation. In conclusion, specific LAB can improve plant-based product fermentation rates, organoleptic properties and thus help to promote plant-based proteins in our diet.

The organoleptic properties of traditional dairy beverages done with non-conventional dairy species (horse, camel), popular in Central Asia, were rarely described in the literature. To characterize the Volatile Organic Compounds (VOC) profile of fermented mare milk, 12 samples of cow milk, used as matrix were inoculated by different strains from two types of bacteria (bacilli and cocci) isolated from natural fermented mare milk. The analysis performed by Gas Chromatography coupled with Solid-Phase Micro-Extraction allowed identification of 160 different compounds from the 12 strains, and 90 from natural fermented mare milk. After cluster analysis, 3 types of profiles were observed. Those profiles were distinct by the amount of acid compounds (low, medium, high), negatively related to aromatic and aliphatic hydrocarbons. The analysis of the mean volatile compound profile of each type of bacteria (bacilli and cocci) by factorial discriminant analysis showed that 3 molecules (oxime-methoxy-phenyl, propanedioic acid propyl and 2-propanamine) allowed to well class 100% of the samples. Further researches on bacterial identification and experiments with different fermentation matrices from other dairy species will be conducted.

Gowe is a very popular traditional fermented and non-alcoholic cereal beverage in Benin. The purpose of the present study was to characterize the physical-chemical properties, nutritional and anti-nutrient composition, microbiological safety and mycotoxin occurrence in the various types of gowe. Six to nine samples from four types of technologies were analyzed for the various aspects of quality. Proximate composition, physical properties (particle size, viscosity), nutritional (amino-acid profile) and anti-nutritional (tannin, phytate, and cyanide levels) properties of gowe resulting from the different traditional processes are quite similar except for the physical properties, the differences of which originate from the raw materials and the diverse cooking conditions. The main hazard of traditional gowe are potential mycotoxin contamination, and presence of cyanogenic compounds, which are linked to raw material contamination and the malting step, and occurrence of E. coli and Enterobacteriacae (gowe is free from other pathogens), linked to inappropriate handling and to the packaging material. The main defects are low α-amylase activity of malt linked to poor control of the malting process, low content of several essential amino-acids in the raw materials and which is deteriorated by the cooking step. Several potential solutions are discussed for paving the way for upgrading and re-engineering this process.

Food spoilage is a major issue for the food industry, leading to important food waste and economic losses. In the context of a growing consumers’ demand for clean label foods [1], antifungal cultures are of growing interest as an alternative to chemical preservatives or a complement tool to hurdle technologies. Our aim was to select effective antifungal cultures in fermented dairy products and to identify the compounds most likely involved in their activity. Lactic acid bacteria and propionibacteria strains were screened alone and in combinations in yogurt and cheese models against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum, and Yarrowia lipolytica). Two binary combinations of lactobacilli were then selected to be evaluated in situ as adjunct cultures in sour cream and semi-hard cheeses produced at the pilot scale, to evaluate i) their antifungal activity during both challenge tests and shelf life tests, and ii) their impact on organoleptic properties. Four chromatography methods were combined to analyse antifungal compounds.Two binary combinations, A1 and A3 composed of L. plantarum L244 associated with either L. harbinensis L172 or L. rhamnosus CIRM-BIA1113, respectively, were selected from the screening step. Both combinations showed antifungal activity during in situ assays, with a higher efficacy of the combination A1 and a higher overall activity in sour cream compared to cheese [2]. In sour cream, A1 delayed the growth of P. commune, M. racemosus and R. mucilaginosafor 2-24 days depending of the antifungal culture inoculum, without effect on organoleptic properties at low inoculum (106colony-forming units (CFU)/mL). In semi-hard cheese, A1 delayed the growth of P. commune for 1 to 6 days. Analyses of antifungal compounds revealed that 33 compounds were in significantly higher amount in at least one product inoculated with an antifungal culture compared to the controls [3]. They were present at concentrations below their minimum inhibitory concentration and thus could act in synergy. Our screening approach allowed developing two antifungal combinations that exhibit significant antifungal activity and provide future prospects for use as bioprotective cultures in dairy products. This extensive study also contributes to improve the knowledge about the action mode of antifungal lactobacilli

Les produits fermentés sont consommés dans le monde entier depuis des siècles. Les végétaux fermentés font l’objet d’un regain d’intérêt, suivant les tendances du végétarisme, de la recherche de naturalité et du développement durable. Le CTCPA et le pôle de compétitivité VEGEPOLYS vous proposent ce séminaire de formation, au cours duquel leurs intervenants feront le point sur les tendances de ce marché, sur l’état de l’art et les limites des connaissances techniques et scientifiques sur le sujet.

Objectifs/ObjectivesThe study aims to improve knowledge over LAB characteristics to ferment a commercial soy juice. Matériels et Méthodes/Materials and methodsFirst 280 strains of LAB from 23 different species of Lactococcus, Streptococcus, Lactobacillus and Leuconostoc genera were tested for their abilities to ferment soy juice containing sucrose, raffinose or stachyose as carbon source and to acidify synthetic media containing sucrose, raffinose or stachyose as unique carbon source. Then, fermented soy juices (FSJ) were produced by 10 h of fermentation (inoculation level 1 %), followed by 14 days of storage at 4 °C. The scents of FSJ were sensorially characterized and FSJ’s primary and secondary metabolites were analyzed. Résultats principaux/Main ResultsWithin 48 h, 39 % of L. lactis strains, 82 % of Lactobacillus strains and all S. thermophilus strains could acidify the synthetic medium with sucrose, as previously described in fermentation patterns of these species. 101 strains representing 18 species could use raffinose and 12 strains representing 6 species could use stachyose. 94 % of the strains that could use sucrose could also ferment soy juice, whose sucrose is the major sugar. Interestingly, 3 strains from species never tested to ferment soy juice, L. kunkeei, L. xiangfangensis and L. pontis revealed abilities to ferment soy juice. Among the 280 strains, 116 strains could acidify soy juice below 6.0 pH units within 10 h. Among the 116 corresponding FSJs, the scents of 48 FSJs were judged as acceptable. The use of sucrose was species-dependent: less residual sucrose was present in FSJs from strains of L. plantarum, L. pentosus, L. lactis, L. delbrueckii and L. coryniformis species compared to S. thermophilus strains. None of the strains used raffinose nor stachyose. S. thermophilus strains produced more lactic acid and less acetic acid than the other strains, in agreement with their homolactic metabolism. Due to acetic acid, FSJs from L. plantarum were more frequently described as ‘sour’. In addition, some strains presented specific traits. For example, CIRM-BIA2115 of L. plantarum produced a floral scent in FSJ likely related to the production of phenylethan-1-ol observed. ConclusionThe present study is an advance on the knowledge of LAB diversity and their capacity to ferment soy juice. It shows that LAB capacity to use sucrose is important to ferment soy juice and highlights the importance of screening due to the diversity of LAB metabolism.

Mucositis, a common and often debilitating side effect of cancer chemotherapy, is a clinically important gastrointestinal inflammatory disease. It consists in a painful inflammation and ulceration of the mucosa lining the digestive tract, which may compromise proper nutrition of the patient, as well as termination of the treatment. It thus increases mortality and morbidity and contributes to rising health care costs. Its treatment is mainly supportive and often fails to relieve symptoms. Several studies indicate a role of the microbiota and suggest a healing effect of probiotic ingestion. Probiotic bacteria may maintain homeostasis and reduce side effects of chemotherapy toxicity. A major limit to probiotic bacteria anti-inflammatory effect is their susceptibility to digestive stresses and several studies indicate the key protective role of food matrices in increasing probiotics’ effect via the protection of bacteria against digestive constraints. In this study, we investigated the role of fermented milk, using Lactobacillus casei or Propionibacterium freudenreichii as probiotic starter, and of its fortification via addition of whey proteins.Matériels et Méthodes/Materials and methodsWe evaluated the role of whey protein isolate (WPI), when added to skim milk fermented by L. casei BL23 or by P. freudenreichii 138, as a protective matrix against in vitro stress challenges. In addition, we investigated in vivo the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). The monitored outcomes included weight loss, ileum histopathological score, villus height, crypt depth and number of mucus-producing goblet cells.Résultats principaux/Main ResultsThis study demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. All symptoms of mucositis were drastically reduced by the consumption of developed probiotic fermented milks.ConclusionThis study evidenced the protective effect of selected strains of both lactic and propionic acid bacteria, in the context of induced mucositis. It confirmed that selected strains might be used bot as starter and probiotic. It opens new avenues for the development of fermented functional foods for target populations.

Artisanal fermented foods represent a cultural heritage that needs to be preserved and protected; this is particularly true in the context of industrial food standardization. The manufacture and unique sensorial properties of such products mainly relies on autochthonous complex microbiota and their activities. The present study focuses on Pélardon, a Protection Designation of Origin (PDO) French artisanal soft rind cheese, exclusively produced from raw goat’s milk using a back slopping method in mountainous areas in southern France. Pélardon microbial ecology and dynamics were studied by culture-dependent and -independent methods. A complete production was followed at an artisanal cheese-making factory and sampling was performed at different cheese-making steps from the raw milk to cheese ripened for 3 months. Different population were monitored on different media. Fungal and bacterial isolates were collected and identified. The achieved results were compared to metabarcoding analyses targeting ITS 2 region for Fungi and V3-V4 region of the 16S RNAr for Bacteria. Focusing on cultivable microorganisms, higher microbial populations were observed in whey used for inoculation compared to raw milk. Results also highlighted the importance of fungi. Despite the initial low inoculum, yeast growth began as early as milk acidification and during the renetting step. Metagenetic results outlined the differences in both fungal and bacterial communities between cheese rind and core samples. Bacterial diversity increased during the ripening specially in the rind compared to the curd stage. Although yeasts were present early in the processing, fungal diversity remained limited at the end of ripening. To better understand the origin of the observed microbial diversity, environmental and raw material samples were also investigated and ongoing work should provide new insights about the dynamics of these microbial communities.

Consumer's demand for naturally preserved food products is growing and the use of bioprotective cultures is an alternative to chemical preservatives or a complementary tool to hurdle technologies to avoid or delay fungal spoilage of dairy products. To develop antifungal cultures for the dairy product biopreservation, experiments were conducted both in vitro and in situ. Firstly, the antifungal activity of 32 strains of lactic acid bacteria (LAB) and propionibacteria was screened alone, and then on combinations based on 5 selected lactobacilli strains. This screening was performed in yogurt and cheese models against four major spoilage fungi previously isolated from contaminated dairy products (Penicillium commune, Mucor racemosus, Galactomyces geotrichum, and Yarrowia lipolytica). Selected combinations were then tested as adjunct cultures in sour cream and semi-hard cheeses produced at a pilot scale to evaluate their antifungal activity during challenge tests against selected fungal targets (P. commune, M. racemosus, and Rhodotorula mucilaginosa) and shelf life tests; and their impact on product organoleptic properties. The screening step allowed selecting two binary combinations, A1 and A3 composed of Lactobacillus plantarum L244 and either Lactobacillus harbinensis L172 or Lactobacillus rhamnosus CIRM-BIA1113, respectively. In situ assays showed that the A1 combination delayed the growth of P. commune, M. racemosus and R. mucilaginosa for 2-24 days on sour cream depending of the antifungal culture inoculum, without effect on organoleptic properties at low inoculum (106 colony-forming units (CFU)/mL). Moreover, the A1 and A3 combinations also delayed the growth of P. commune in semi-hard cheese for 1-6 days and 1 day, respectively. Antifungal cultures neither impacted the growth of starter cultures in both sour cream and cheese nor the products' pH, although post acidification was observed in sour cream supplemented with these combinations at the highest concentrations (2.107 CFU/mL). The combination of both in vitro and in situ screening assays allowed developing 2 antifungal combinations exhibiting significant antifungal activity and providing future prospects for use as bioprotective cultures in dairy products.

Objectifs Le microbiote intestinal humain est un écosystème complexe et dynamique. Il exerce de nombreuses fonctions biologiques et métaboliques. Le maintien de son équilibre (composition, activité) est important pour la santé et le bien-être de l’hôte et peut être influencé par de nombreux facteurs, comme l’alimentation. Les produits laitiers fermentés, qui contiennent une grande quantité de bactéries lactiques vivantes, peuvent contribuer à cet équilibre mais la compréhension de leur impact reste encore limitée. Ce travail a pour objectif de mieux comprendre, à partir de l’utilisation d’outils métaomiques, l’impact de la consommation de lait fermenté et de bactéries lactiques vivantes sur la composition et l’activité du microbiote intestinal. De plus, il vise à évaluer l’état physiologique et métabolique des bactéries lactiques en transit dans le tractus digestif. Matériels et Méthodes Trois groupes de 8 souris à flore humanisée (ie transplantation d’un microbiote fécal d’un individu sain à des souris axéniques) ont été alimentées avec (i) un régime contrôle sans lait (alimentation traditionnelle des souris + tampon PBS), (ii) un régime avec lait fermenté (alimentation traditionnelle des souris + un lait fermenté par les 2 bactéries du yaourt, Streptococcus thermophilus et Lactobacillus bulgaricus), ou (iii) un régime avec lait fermenté thermisé (alimentation traditionnelle des souris + un lait fermenté par les 2 bactéries du yaourt puis thermisé à 95°C pendant 25 min). La composition et l’activité du microbiote humain transplanté chez les souris ont été évaluées respectivement par métagénomique shotgun et métatranscriptomique à partir d’échantillons fécaux. Résultats principaux L’analyse quantitative et le suivi de l’activité du microbiote intestinal de souris à flore humanisée est réalisable grâce à des approches métaomiques. La consommation de lait fermenté (thermisé ou pas) ne modifie pas la composition du microbiote intestinal humain chez la souris mais a un impact significatif sur son activité. L’expression des gènes est modifiée pour la plupart des genres bactériens dominants, et modulant par conséquent leurs fonctions. Cet impact est d’autant plus important que la consommation de lait fermenté est régulière et prolongée dans le temps. En revanche, il n’a pas été possible de suivre l’activité des bactéries lactiques en transit qui sont minoritaires. Conclusion Ce travail met en évidence l’intérêt des méthodes métaomiques pour suivre l’impact de l’alimentation et ouvre des perspectives sur l’exploration des effets bénéfiques potentiels des laits fermentés sur le microbiote intestinal humain.

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

Microbial approaches againts bovine mastitis in dairy cows. STLOpendays

Promoting cooperation between lactic acid bacteria within a community: towards new food applications. STLOpendays

Food spoilage is a major issue for the food industry, leading to important food waste and economic losses. In the context of a growing consumers’ demand for clean label foods [1], antifungal cultures are of growing interest as an alternative to chemical preservatives or a complement tool to hurdle technologies. Our aim was to select effective antifungal cultures in fermented dairy products and to identify the compounds most likely involved in their activity. Lactic acid bacteria and propionibacteria strains were screened alone and in combinations in yogurt and cheese models against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum, and Yarrowia lipolytica). Two binary combinations of lactobacilli were then selected to be evaluated in situ as adjunct cultures in sour cream and semi-hard cheeses produced at the pilot scale, to evaluate i) their antifungal activity during both challenge tests and shelf life tests, and ii) their impact on organoleptic properties. Four chromatography methods were combined to analyse antifungal compounds.Two binary combinations, A1 and A3 composed of L. plantarum L244 associated with either L. harbinensis L172 or L. rhamnosus CIRM-BIA1113, respectively, were selected from the screening step. Both combinations showed antifungal activity during in situ assays, with a higher efficacy of the combination A1 and a higher overall activity in sour cream compared to cheese [2]. In sour cream, A1 delayed the growth of P. commune, M. racemosus and R. mucilaginosafor 2-24 days depending of the antifungal culture inoculum, without effect on organoleptic properties at low inoculum (106colony-forming units (CFU)/mL). In semi-hard cheese, A1 delayed the growth of P. commune for 1 to 6 days. Analyses of antifungal compounds revealed that 33 compounds were in significantly higher amount in at least one product inoculated with an antifungal culture compared to the controls [3]. They were present at concentrations below their minimum inhibitory concentration and thus could act in synergy. Our screening approach allowed developing two antifungal combinations that exhibit significant antifungal activity and provide future prospects for use as bioprotective cultures in dairy products. This extensive study also contributes to improve the knowledge about the action mode of antifungal lactobacilli

Le procédé alimentaire From’Innov permet de produire des fromages selon un protocole ne nécessitant ni égouttage ni affinage. Son concept repose sur l’assemblage de deux matrices laitières : une matrice de texture et une matrice aromatique, constituée d’une ou plusieurs cultures d’au moins un microorganisme producteur d’arôme. L’objectif de cette étude était d’optimiser la production des arômes dans cette matrice. Quatre espèces ont été cultivées dans différentes conditions : Propionibacterium freudenreichii et Propionibacterium acidipropionici pour le propionate, Hafnia alvei pour les composés soufrés et Yarrowia lipolytica pour les acides gras libres. Les composés d’arôme ont été analysés en HPLC pour les acides et en CPG-SM pour les composés volatils. Leur teneur a été augmentée dans les matrices aromatiques. Des fromages ont été produits à partir des matrices les plus concentrées, mais seulement celui incorporé avec 5% de matrice aromatique de Y. lipolytica a été discerné par évaluation sensorielle. D’autres pistes pour optimiser et diversifier l’aromatisation sont en cours d’étude.

Fermented cereals are part of the main traditional diets of many people in Africa, usually obtained from artisanal production. The intensification of their manufacturing, responding to the consumers demand, requires a better control to ensure their sanitary, nutritional, and taste qualities, hence, the need of selecting accurate and safe starter cultures. In the present study, 48 lactic acid bacteria (LAB) strains, previously isolated from Algerian fermented wheat lemzeiet, were analyzed for different technological properties. 14 LAB strains, belonging to Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Lactobacillus brevis, and Leuconostoc mesenteroides species, decreased rapidly the pH of the flour extract broth close to 4 or below. 91% of strains showed extracellular protease activity, but only 12% were amylolytics. 18 LAB strains inhibited or postponed the growth of three fungal targets Rhodotorula mucilaginosa UBOCC-A-216004, Penicillium verrucosum UBOCC-A-109221, and Aspergillus flavus UBOCC-A-106028. The strains belonging to Lactobacillus spp., Leuconostoc fallax, L. mesenteroides, and Weissella paramesenteroides were the most antifungal ones. Multiplex PCR for biogenic amines' production did not reveal any of the genes involved in the production of putrescine, histamine, and tyramine for 17 of the 48 strains. The obtained results provided several candidates for use as starter culture in the future production of lemzeiet.

Trabajo presentado en el VII Congrès International de Biotechnologie et Valorisation des Bio-Ressources, celebrado en Tabarka (Túnez), del 20 al 23 de marzo de 2019, Au cours des dernières décennies, une plus grande attention est portée à la sécurité alimentaire afin de protéger la santé humaine que ce soit sur les contaminants chimiques, contaminants physiques ou contaminants d¿origine biologique. Le secteur des aliments fermentés nécessite une attention particulière en raison de la complexité de leur processus de transformation biotechnologique. La recherche de contaminants d¿origine naturelle et biologique tels que les amines biogènes est particulièrement importante, car certaines de ces molécules, notamment l¿histamine, sont dangereuses pour l¿homme si la dose ingérée est trop élevée. L¿objectif de cette étude consiste en la détéction des souches de bactéries lactiques spécifiques (Lactobacillus, Lactococcus et Enterococcus) productrices d¿amines biogènes ainsi que la recherche des gènes codant pour les enzymes impliquées dans la production de deux amines biogènes (Tyramine, Histamine) avec des amorces spécifiques de chaque amine biogène étudié par PCR ainsi qu¿une étude sur milieu de culture spécifique. Les bactéries lactiques ont notamment été identifiées à partir_de l¿ARNr16S.

Lactobacillus, Pediococcus, Oenococcus and Leuconostoc are the main Lactic Acid Bacteria (LAB) genera present in cider as they are able to survive this hostile environment. LAB play a significant role in cider quality, for example in the process of malolactic fermentation, even though they can also be involved in spoilage of cider (production of biogenic amines, exopolysaccharides, off-flavours…). In this context a better monitoring of the fermentation process is a matter of interest to guarantee cider quality. In the present study, we designed a genus-specific multiplex PCR for a rapid and simultaneous detection of the four main LAB genera involved in cider production. This multiplex PCR worked equally with purified genomic DNA of bacterial isolates and with colonies directly picked from agar plates. This new PCR method was also successfully extended to wine and dairy isolates, and thus constitutes an effective tool to quickly identify LAB associated with fermented foods. Moreover, many biodiversity studies would also benefit from this fast, cheap and reliable identification method.

Prodinra 463822; Fungal contamination of dairy products is responsible for economic losses and food waste. In a context of “preservative-free” product demand, bioprotective cultures and their metabolites represe,t an alternative of interest of chemical preservatives to control these spoilers.The objective of this study was i) to select microorganisms exhibiting an antifungal activity, in order to elaborate bioprotectivecultures applicable in dairy products, and ii) to study the compounds potentially supporting the observed activity. Firstly, the antifungal activity of 32 strains of lactic acid and propionic bacteria screened in cheese model and yogurt. Strain combinaison study and safety assessment led to the selection of 2 binary lactobacilli combinations (A1 and A3). Their efficiency and applicability were then evaluated in pilot-scale productions of sour cream and cheese.Challenge and shelf life tests showed that depending on the dairy product, A1 and A3 have a similar or higher antifungal activity than the commercial bioprotective cultures. In addition, depending of inoculum, A1 and A3 did not impact the technological and organoleptic characteristics. Chromatographic methods and statistical analyses allowed identifying cocktails of 2 to 17 compounds, according to the considered dairy product and culture that probably support the antifungal activity.The obtained results contribute to a better understanding of the antifungal activity action mechanisms and should lead to the development of antifungal cultures to replace preservatives in dairy products.; La contamination fongique des produits laitiers est à l’origine de pertes économiques conséquentes et de gaspillage alimentaire. Dans un contexte de demande pour plus de « naturalité », les cultures de bioprotection et leurs métabolites représentent une alternative d’intérêt aux conservateurs chimiques pour lutter contre ces contaminants.Les objectifs de cette thèse étaient i) de sélectionner des micro-organismes présentant une activité antifongique, pour élaborer des cultures de bioprotection applicables dans des produits laitiers, et ii) d’étudier les composés potentiellement supports de l’activité antifongique observée. Dans un premier temps, l’activité antifongique de 32 souches de bactéries lactiques et propioniques a été étudiée en modèles « fromage » et « yaourt ». L’étude de combinaisons de souches et de leur innocuité a conduit à sélectionner 2 combinaisons binaires de lactobacilles (A1 et A3). Leur efficacité et applicabilité a été évaluée à l’échelle pilote en fabrication de crème fraîche et de fromage.Les challenges tests et tests d’usages ont montré que selon le produit laitier, A1 et A3 ont une activité antifongique similaire ou supérieure que les cultures bioprotectrices commerciales. Selon l’inoculum ajouté, ces cultures n’impactent pas les caractéristiques technologiques et organoleptiques des produits laitiers. Des méthodes chromatographiques des composés antifongiques suivies d’analyses statistiques ont permis de mettre en évidence des « cocktails » de 2 à 17 composés, selon la matrice et la culture considérée, qui sont probablement supports de l’activité antifongique.Ces travaux contribuent à une meilleure compréhension des mécanismes d’action de l’activité antifongique et devraient conduire au développement de cultures antifongique pour remplacer les conservateurs dans les produits laitiers.

In the context of a demand for “preservative-free” food products, biopreservation appears as a promising alternative to either replace or reduce the use of chemical preservatives. The purpose of this study was to evaluate the antifungal activity of a collection of lactic acid bacteria (n = 194), and then to evaluate the applicability and efficacy of selected ones used as bioprotective cultures against mold spoilers in dairy and bakery products. First, lactic acid bacteria were isolated from various Algerian raw milk samples and Amoredj, a traditional fermented product. Secondly, in vitro screening tests against Mucor racemosus UBOCC-A-109155, Penicillium commune UBOCC-A-116003, Yarrowia lipolytica UBOCC-A-216006, Aspergillus tubingensis AN, Aspergillus flavus T5 and Paecilomyces formosus AT allowed for the selection of 3 active strains, namely Lactobacillus plantarum CH1, Lactobacillus paracasei B20 and Leuconostoc mesenteroides L1. In situ tests were then performed to validate their activity in actual products (sour cream and sourdough bread) challenged with fungal spoilers. These tests showed that antifungal LAB could slow the fungal target growth and could be candidates of interest for industrial applications. Finally, organic acids and various antifungal compounds produced in sour cream and sourdough bread by the selected LAB, and thus potentially supporting the observed antifungal activity, were identified and quantified by HPLC and LC-QTOF.

Natural sourdoughs are commonly used in bread-making processes, especially for organic bread. Despite its role in bread flavor and dough rise, the stability of the sourdough microbial community during and between bread-making processes is debated. We investigated the dynamics of lactic acid bacteria (LAB) and yeast communities in traditional organic sourdoughs of five French bakeries during the bread-making process and several months apart using classical and molecular microbiology techniques. Sourdoughs were sampled at four steps of the bread-making process with repetition. The analysis of microbial density over 68 sourdough/dough samples revealed that both LAB and yeast counts changed along the bread-making process and between bread-making runs. The species composition was less variable. A total of six LAB and nine yeast species was identified from 520 and 1675 isolates, respectively. The dominant LAB species was Lactobacillus sanfranciscensis, found for all bakeries and each bread-making run. The dominant yeast species changed only once between bread-making processes but differed between bakeries. They mostly belonged to the Kazachstania clade. Overall, this study highlights the change of population density within the bread-making process and between bread-making runs and the relative stability of the sourdough species community during bread-making process.

International audience; Fungi are commonly identified as the cause for dairy food spoilage. This can lead to substantial economic losses for the dairy industry as well as consumer dissatisfaction. In this context, biopreservation of fermented dairy products using lactic acid bacteria, propionibacteria and fungi capable of producing a large range of antifungal metabolites is of major interest. In a previous study, extensive screening was performed in vitro and in situ to select 3 dairy fermentates (derived from Acidipropionibacterium jensenii CIRM-BIA1774, Lactobacillus rhamnosus CIRM-BIA1952 and Mucor lanceolatus UBOCC-A-109193, respectively) with antifungal activity. The aim of the present study was to determine the main compounds responsible for this antifungal activity. Fifty-six known antifungal compounds as well as volatiles were targeted using different analytical methods (conventional LC and GC, GC–MS, LC-QToF). The most abundant antifungal compounds in P. jensenii-, L. rhamnosus- and M. lanceolatus-derived fermentates corresponded to propionic and acetic acids, lactic and acetic acids, and butyric acid,respectively. Many other antifungal compounds (organic acids, free fatty acids, volatile compounds) were identified but at lower levels. In addition, an untargeted approach using nano LC-MS/MS identified a 9-amino acid peptide derived from αs2-casein in the L. rhamnosus-derived fermentate. This peptide inhibited M. racemosus and R. mucilaginosa in vitro. This study provides new insights on the molecules involved in antifungal activities of food-grade microorganism fermentates which could be used as antifungal ingredients in the dairy industry.

Folates represent an essential vitamin in the human diet at all ages, particularly during pregnancy and infancy, as it is required for the production of new cells. In many African countries, the main staple foods are based on cereals, which are always consumed after processing. Fermentation is one of the processing, which could increase folate contents in foods. The objective of this work was to increase folates intake of African people through the consumption of cereal-based fermented foods using fermentation. Seven types of cereal-based fermented foods (CBFF), commonly consumed in West Africa, were investigated in this study. Total folate content of cereal-based fermented ranged between 1.8 and 31.3 µg/100g fresh weight, and was almost always lower than in the raw material (13.8-73.4 µg/100g fresh weight). Folate losses occurred during some processing steps like debranning, soaking and drying steps. However, fermentation was able to increase the folate content in some CBFF. Folate bioaccessibility was assessed using a static in vitro digestion model, and ranged from 23% to 81%. The bioaccessible folate content was influenced by total folate content, the structure of food matrices that modulated folate release, and folate stability during digestion process. Calculations of the contributions of CBFF to the reference nutrient intake for folate showed that folate intakes from these foods would cover a maximum of 8% of the folate requirements for young children. Porridges prepared with starter cultures of lactic acid bacteria selected for the nutritional properties (folate synthesis, starch hydrolysis) had significantly higher folate contents (up to 8.7 µg/100 g fresh matter) than the porridge prepared using the traditional process (2.5-5.4 µg/100 g fresh matter). Back slopping using an inoculum from a spontaneous fermentation also enabled an interesting increase in folate contents (up to 7.4 µg/100 g fresh matter). The bacterial diversity of seven cereal-based fermented foods from Burkina Faso, Ethiopia and Finland were assessed using 16S rRNA amplicon sequencing. Lactic acid bacteria genus, including Lactobacillus, Enterococcus, Weissella, Pediococcus, Lactococcus and Streptococcus were the main bacteria present in cereal-based fermented foods. Several potentially pathogenic bacteria, namely, Bacillus, Pseudomonas, Staphylococcus, Erwinia, Escherichia, Klebsiella and Acinetobacter were also found in some intermediary products resulting from storage and wet milling. These microorganisms were reduced by fermentation and finally removed after the cooking step.; Les folates sont des vitamines indispensables à tous les âges, particulièrement pendant la grossesse et l’enfance, étant donné leur fonction dans la division cellulaire. Le régime alimentaire en Afrique est essentiellement basé sur les céréales, consommées toujours après transformation. La fermentation est l’un des moyens de transformation des céréales pouvant augmenter les folates dans les aliments. L’objectif de ce travail était d’utiliser la fermentation pour augmenter les ingérés en folates des populations africaines via la consommation d’aliments céréaliers fermentés. Sept aliments céréaliers fermentés couramment consommés en Afrique de l’Ouest ont été investigués. La plupart des aliments avaient des teneurs en folates (1,8–31,3 µg/100g matière fraîche) inférieures à celles des céréales de départ (13,8-73,4 µg/100g matière fraîche). Des pertes en folates ont lieu au cours de certaines étapes de procédés dont le décorticage, le séchage, le trempage, le broyage et la filtration. Toutefois, la fermentation a permis une augmentation de la teneur en folates dans certains aliments. La bioaccessibilité des folates, évaluée à l'aide d'un modèle de digestion statique in vitro, variait de 23% à 81%. Elle était influencée par la matrice alimentaire et la stabilité des folates au cours de la digestion. Il a été calculé qu’au maximum 8% des besoins journaliers en folates des jeunes enfants consommant l’un des aliments étudiés pourraient être couverts. Des essais d’inoculation de bouillies fermentés à base de mil avec des souches de bactéries lactiques sélectionnées pour leurs propriétés nutritionnelles (synthèse de folates, hydrolyse de l’amidon) permettaient d’augmenter significativement les teneurs en folates (jusqu’à 8,7 µg/100 g matière fraîche) par rapport à leur équivalent préparées de manière traditionnelle (2,5-5,4 µg/100 g matière fraîche). L’inoculation par un pied-de-cuve provenant d’une fermentation spontanée permettait aussi une augmentation significative des teneurs folates (jusqu’à 7,4 µg/100 g matière fraîche). La caractérisation de la diversité bactérienne de 7 aliments céréaliers fermentés du Burkina Faso, d’Ethiopie et de la Finlande montrait que les bactéries lactiques du genre Lactobacillus, Enterococcus, Weissella, Pediococcus, Lactococcus et Streptococcus étaient les principaux acteurs de la fermentation de ces aliments. Toutefois, une présence non négligeable d’autres microorganismes potentiellement pathogènes des genres Bacillus, Pseudomonas, Staphylococcus, Erwinia, Klebsiella, Escherichia et Acinetobacter a été identifiée. Cette contamination était liée à certaines étapes du procédé de transformation des céréales dont le stockage et broyage dans les moulins publics. Ces microorganismes pathogènes étaient réduits par fermentation et finalement éliminés après l'étape de cuisson.

Fungi are commonly responsible for dairy product spoilage, which leads to substantial economic losses for the dairy industry as well as consumer dissatisfaction. Lactic acid bacteria can play an active role in the bioprotection of fermented dairy product, by producing a large range of antifungal metabolites. In a previous study, 3 binary combinations of lactobacilli strains were selected from a screening of the antifungal activity of 32 strains tested as adjunct cultures in yogurt and cheese models and the applicability of 2 of them in dairy products was validated at the pilot scale in sour cream and semi-hard cheese. Thus, the aim of the present study was to investigate the compounds produced by these antifungal cultures in these dairy products with a potential role in the observed antifungal activity. Using four different analytical methods including high performance liquid chromatography and gas chromatography combined or not with mass spectrometry, 53 compounds with known antifungal activity were detected in at least one of the four dairy products analyzed. Each antifungal culture produced a cocktail of 2 to 27 compounds, depending on the product. Acetic acid, diacetyl, hydroxyphenyllactic acid, pyruvic acid, phenyllactic acid, succinic acid, decanoic acid and some long-chain unsaturated fatty acids were the most commonly identified compounds. Their concentrations markedly differed according to the considered dairy product. Lactic, acetic, and citric acids were the most abundant compounds, while most of the other compounds were present in concentrations

INTRODUCTION Propionibacterium freudenreichii is an emergent probiotic bacterium, belonging to dairy propionibacteria, within the group of Actinomycetales, and possessing the GRAS (Generally Recognized As Safe, USA, FDA) and QPS (Qualified Presumption of Safety, EFSA, European Union) status. It is a traditional Swiss-type cheeses starter and an emergent probiotic, exerting several beneficial effects, including anti-inflammatory modulation of gut inflammation. This feature relies on several metabolites and on surface proteins including in particular the surface protein SlpB. METHOD In the present study, we assessed first the impact of proteolytic degradation of SlpB on its immunomodulatory effect on colon epithelial HT-29 cells. Intact P. freudenreichii cells, purified surface layer proteins (SLPs), as well as trypsin-proteolyzed SLPs, were used to stimulate HT-29 cells, in the presence or absence of pro-inflamatory LPS. Cytokines were then quantified to evaluate the inflammatory response. We then investigated the potential of cheese matrix as a delivery vehicle of anti-inflammatory P. freudenreichii MAMP to the colon, by comparing dairy liquids matrices to a solid Swiss-type cheese matrix, using two in vitro digestion models. Integrity of SlpB was monitored using western blotting. RESULTS In this study, we firstly investigated the relevance to avoid SlpB digestive proteolysis, by comparing the effect of i) P. freudenreichii CIRM-BIA 129, ii) its native SLPs, or iii) peptides resulting from SLPs stimulations, with respect to modulation of HT-29 cells response to lipopolysaccharide (LPS) challenge. The anti-inflammatory effect exerted by P. freudenreichii CIRM-BIA 129 and by native surface proteins (SLPs) on HT-29 cells was abolished by digestive proteolysis. This result confirmed the importance to protect immunomodulatory surface proteins from digestive proteolysis in order to allow gut immune system modulation. Thus, we examined the effect of dairy matrices on P. freudenreichii viability and on SlpB integrity during digestion. In comparison with liquid matrices, the cheese matrix provided an enhanced tolerance to digestive stresses and protection of SlpB towards proteolysis, during two in vitro digestion models: static and dynamic. CONCLUSION These in vitro results provide new insights into the matrix effect on P. freudenreichii probiotic functionalities. The cheese matrix offers significant protection against the digestion stresses by enhancing survival, and by protecting surface layer proteins from proteolysis. However, in vivo investigations are necessary to study the matrix effect during digestion, to follow SLPs expression and to evaluate the matrix effect on immunomodulation by P. freudenreichii within the gut. Such data accordingly will allow the development of new functional foods for the delivery of P. freudenreichii to the gastrointestinal tract of humans in the aim to help prevention or treatment of life-style related diseases with an inflammatory component.

Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese-ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favoured by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. We thus investigated the involvement of a specific propionibacterial surface protein in adhesion of the probiotic Propionibacterium freudenreichii to host cells. We compared several strains of Propionibacterium freudenreichii, with respect to adhesion to cultured human intestinal epithelial cells, using a well-established model. We inactivated genes using a previously developed method, based on the integration of a non-replicative plasmid, harbouring a portion of the target gene. We analysed adhesion of the wild type and the mutant strains using fluorescent-assisted flow cytometry and confocal microscopy. We also sought inhibition of adhesion using antibodies directed against surface layer proteins. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species.