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International audience; The transition towards a more sustainable food system requires that small and mid-sized enterprises increase their economic competitiveness and resilience and strengthen their innovation capacity. However, their specific needs are often neglected, especially in innovation processes that focus on large and expensive improvements.The H2020 project FAIRCHAIN (https://www.fairchain-h2020.eu/) proposes development and adaptation of innovations that strenghen the position of small and mid-sized farmers and processors in the food chains and allow the scale-up and expansion of the production of nutritious food at a regional level. This talk will present some technical and technological innovations devoted to SMEs and producers which may increase food sustainability: as examples the development of an innovative healthy fermented whey-based drink to upgrade the value of whey, a co-product of cheese manufacture ; the development of an innovative packaging machine for liquid or viscous food products in the dairy and fruit & vegetables and dairy sectors, using green or sustainable packaging materials and designed to fulfil hygienic requirements ; or the development of the blockchain technology to improve the traceability , transparence and information sharing, that is of major importance when increasing the number of intermediaries in the food chain.The main challenges in adopting these innovations have been identified thanks to a co-creation process and assessment framework. This talk will discuss these challenges, as well as supporting and hindering factors for putting innovations into practice and possible solutions for their successful implementation

International audience; The FAIRCHAIN project will chair a special session dedicated to innovation for small and medium food producers and enterprises at the 36 edition of the EFFoST International Conference.The presentations will focus on technical and technological innovations specifically geared at SMEs developed and demonstrated by different EU-funded projects as well as deployment challenges and supporting factors.

Aim: Cheese whey is the watery part obtained after milk curdling in cheese-making. Because it contains high quality proteins, whey is mainly valorized by industrial processed to obtain value-added food ingredients. However, these processes are only applicable to some types of whey, are energy-consuming and only concern large-scale dairy plants. As part of FAIRCHAIN project, we aimed at developing whey-based beverages using fermentation as alternative whey valorization process. This study explores the ability of lactic acid bacteria (LAB) to ferment different types of whey, to obtain the best aroma profiles for the beverages.Method: Two types of whey were assessed: a sweet whey from Morbier cheese and an acid one from Comté cheese. 125 LAB strains belonging to 27 species, were selected for their capacity to utilize lactose and their GRAS status. They were evaluated for their ability to ferment both wheys. After fermentation, the products were assessed for their acidification and sensory properties (tastes and sniffing). Sugar content and volatile profile of fermented wheys were evaluated by HPLC and GC-MS respectively.Results: Fermentation outcomes differed according to the whey type: 34 strains did not acidify sweet whey, while 91 strains did not acidify the acid one. For a given whey, acidification was species-dependent: Lactobacillus helveticus and Lactobacillus delbrueckii strains showed the best acidification capacities in both wheys although acidification was higher on sweet whey compared to the acid one. Sensory profile analysis led to different results and were strain-dependent. Regarding sweet whey, 40 strains (12.5%) encompassing 12 species showed a promising aroma profile while only 20 belonging to 7 species were retained for acid whey. Both criteria considered, 26% strains tested on sweet whey showed a good acidification ability and an aroma profile deemed to be acceptable, whereas only 20 strains were retained for Comté acid whey. Noteworthy, only 6 strains showed promising results on both wheys.Conclusion: This study highlighted the huge diversity of the metabolic profiles of LAB strains when used for fermentation of different whey types. Fermentation by well selected bacteria LAB thus appears as a sustainable and an inexpensive process, to valorize whey.

Ces dernières années, les approches métagénomiques (basées sur le séquençage de l’ADN microbien) sont devenues incontournables dans l’étude des écosystèmes microbiens des aliments, supplantant parfois les approches culturales classiques souvent considérées comme laborieuses. A travers l’exemple du Pélardon, fromage artisanal au lait cru de chèvre fabriqué par backslopping de lactosérum, nous avons comparé les communautés microbiennes au cours de la fabrication du fromage révélées par deux approches cuture-indépendantes, i.e. métagénétique 16S et métagénomique Shotgun, ainsi que par approche culturale. Cette dernière a reposé sur la déréplication et l’identification par spectrométrie de masse MALDI-TOF de plus de 600 isolats bactériens isolés de quatre milieux de culture différents. Pour ces trois méthodes, les analyses ont été réalisées sur des échantillons de Pélardon prélevés à trois stades de fabrication : caillage, affinage précoce (2 semaines) et affinage prolongé (2 mois) en analysant croûte et cœur séparément. La reconstruction des dynamiques microbiennes a montré une diversité croissante au cours de la fabrication. Les trois approches s’accordent sur la dominance marquée de Lactococcus lactis dans le caillé, suivi de Leuconostoc mesenteroides. En revanche, les stades d’affinage ont été marqués par de fortes divergences entre les différentes approches, menant à des visions très différentes des communautés, que ce soit en surface ou au cœur du Pélardon. Ainsi, cinq genres parmi lesquels Lacticaseibacillus et Enterococcus, n’ont été détectés que par l’analyse culture-dépendante tandis que six autres genres (i.e. Brachybacterium, Acinetobacter) n’ont été mis en évidence qu’à travers les approches culture-indépendantes. Si certains genres correspondaient à des populations sous-dominantes, il n’en est pas de même pour Lacticaseibacillus paracasei. Cette espèce représentait près de la totalité des flores lactiques cultivables en cœur des fromages affinés et n’a pas été détectée par l’analyse métagénétique ou Shotgun. A l’inverse, sur la base des analyses culture-indépendantes, L. lactis restait dominante tout au long de l’affinage en surface et au cœur, tandis qu’elle n’était plus détectée sur aucun des milieux utilisés par approche-culture dépendante au second stade d’affinage étudié (2 mois). Plusieurs hypothèses biologiques ont été envisagées pour expliquer cette divergence. Toutefois, ces résultats ont permis de mettre en exergue les forces et les biais liées aux approches basées sur l’ADN et illustrent, par ailleurs, la nécessité d’une approche polyphasique dans l’étude des dynamiques microbiennes des fromages artisanaux.

International audience; Le Pélardon est un petit fromage traditionnel au lait cru de chèvre, se consommant frais (14 jours) ou affiné (jusqu’à 3 mois d’affinage), et labellisé AOP depuis 2000. Il est uniquement produit par des producteurs-éleveurs, dans la région montagneuse des Cévennes (sud de la France). Le Pélardon est préparé avec du lait cru de chèvre, qui est ensemencé uniquement par backslopping à partir du lactosérum d’une précédente fabrication et sans recours à des ferments commerciaux. Les flores naturellement présentes dans l’environnement et les matières premières sont donc susceptibles de jouer un rôle prépondérant dans sa fabrication. Ce travail avait pour objectif d’étudier les communautés microbiennes du Pélardon au cours de sa fabrication et de l’affinage et d’en identifier les espèces susceptibles de contribuer à ses caractéristiques finales. Pour cela, des échantillons ont été prélevés à sept stades de fabrication allant du lait cru jusqu’à trois mois d’affinage. Les communautés microbiennes de ces échantillons ont été analysées par méthodes i) culture-dépendante (suivi des populations sur des milieux de culture spécifiques) et identification à l’espèce de 2877 isolats par analyse MALDI-TOF) et ii) culture-indépendante : analyse metagénétique en ciblant les régions ITS2 (levures / champignons) et V3-V4 de l’ADNr 16S (bactéries). Ces dynamiques ont ensuite été corrélées à l’évolution des composés d’arômes (acides et composés volatils) aux mêmes stades de fabrication. En parallèle, l’environnement de production des fromages a été analysé, pour déterminer dans quelle mesure il pouvait être source de micro-organismes. Les résultats ont montré que le Pélardon se caractérisait par une forte diversité bactérienne marquée par une succession d’espèces différentes à chaque stade. En comparaison, la diversité fongique associée au Pélardon s’est avérée faible avec Geotrichum candidum comme espèce dominante. L’analyse de corrélation menée à partir des données biochimiques et microbiennes ont fait ressortir certains microorganismes : quatre espèces bactériennes (Lactococcus lactis, Leuconostoc mesenteroides, Lacticaseibacillus paracasei et Enterococcus faecalis) et trois espèces fongiques (G. candidum, Penicillium commune et Scopulariopsis brevicaulis) au cours de la fabrication et de l’affinage. L. lactis a été identifiée comme responsable de l’acidification en raison de sa forte corrélation à l’acide lactique mais également à l’acétoïne. G. candidum et L. mesenteroides, principalement dominants durant l’étape de séchage, étaient corrélés à des composés volatils issus du catabolisme des acides aminés. Les plus fortes corrélations avec les cétones et les esters, correspondant aux composés les plus abondants aux stades avancés d’affinage ont été observés pour S. brevicaulis et L. paracasei et dans une moindre mesure P. commune et E. faecalis. L’analyse des vecteurs microbiens de l’environnement de production a souligné le rôle du lactosérum comme source des bactéries d’acidification mais aussi de micro-organismes d’affinage précoce. Les espèces contribuant à l’affinage tardif étaient à l’inverse, absentes des matières premières et retrouvées sur certaines surfaces indiquant une origine environnementale. Cette étude a permis de mettre en évidence la diversité microbienne du Pélardon et d’identifier les micro-organismes clés de la fabrication du Pélardon. Les résultats soulignent également le rôle des matières premières mais également l’importance de l’environnement de 10 production comme vecteur de micro-organismes contribuant à la typicité des fromages artisanaux.

International audience; The formation of cheese flavor mainly results from the production of volatile compounds by microorganisms. We investigated how fine-tuning cheese-making process parameters changed the cheese volatilome in a semi-hard cheese inoculated with Lactococcus (L.) lactis, Lactiplantibacillus (L.) plantarum, and Propionibacterium (P.) freudenreichii. A standard (Std) cheese was compared with three variants of technological itineraries: a shorter salting time (7 h vs 10 h, Salt7h), a shorter stirring time (15 min vs 30 min, Stir15min), or a higher ripening temperature (16°C vs 13°C, Rip16°C). Bacterial counts were similar in the four cheese types, except for a 1.4 log 10 reduction of L. lactis counts in Rip16°C cheeses after 7 weeks of ripening. Compared to Std, Stir15min and Rip16°C increased propionibacterial activity, causing higher concentrations of acetic, succinic, and propanoic acids and lower levels of lactic acid. Rip16°C accelerated secondary proteolysis and volatile production. We thus demonstrated that fine-tuning process parameters could modulate the cheese volatilome by influencing specific bacterial metabolisms.

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.

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.

International audience

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.

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.

1. Introduction Fermented foods are complex biological ecosystems, harbouring diverse microbial communities which contribute to the quality of these food products. In most industrial processes, these communities are advisedly chosen as starter culture for their adaptation to: (i) the substrates to be fermented (milk, vegetable) (ii) the process (°C, O2, NaCl) and (iii) the desired overall quality of the food product in terms of texture, flavour, nutrition and health benefits. For this purpose, technological properties have been mainly assessed by a time-consuming in vitro screening. We hypothesis that the huge amount of bibliographic and microbial genomic data available in public databases would enable an in silico preselection of the strains of interest before wet laboratory screening experiments. 2. Objectives Our aim was to provide (i) adapted bioinformatic tools to mine the diversity of microbial technological properties and metabolisms (ii) a strategy to preselect strains based on their phenotypes and on their genetic potential (sugar utilization, aroma production). 3. Material and Methods We used a bioanalysis approach to provide insight into the presence of enzyme-encoding genes responsible for targeted technological properties. KEGG, Metacyc, NCBI and CAZY databases were queried by a search on enzyme numbers, names and sequence alignments. We developed Florilege (http://migale.jouy.inra.fr/Florilege/), a database of microbial phenotypes based on text-mining to gather microbial growth conditions. 4. Results Combining both approaches, we set up a bioinformatic preselection of lactic acid bacteria able to degrade sugar from milk, cereal or legume based juice to develop yogurt-like products. Database queries provided a list of microbial species with genes encoding key targeted enzymes. A set of preselected anaerobic, mesophilic and thermophilic strains has been successfully screened on different milk and juice in our wet laboratory providing potential new starter strains. 5. Conclusion The developed tools and the strategy can be usefully applied to other domains like bioremediation and white biotechnology.

Pélardon is a Protection Designation of Origin (PDO) French artisanal soft rind cheese, produced in mountainous areas in southern France. It is exclusively produced from raw goat’s milk using a back slopping method for inoculation. Its manufacture and unique organoleptic properties rely on the autochthonous microbiota and their activities. The aim of this work is thus to study Pélardon microbial (fungal & bacterial) ecology and dynamics during cheese-making process by culture-dependent and -independent approaches.

Propionibacterium freudenreichii (PF) is a food grade bacterium with “generally recognized as safe” status, most commonly used as a starter for the manufacture of fermented dairy products such as Emmental and Leerdammer cheeses. It is also known for its production of vitamin B12 and propionic acid, and is studied for its remarkable anti-inflammatory properties[1,2]. Exopolysaccharide (EPS) production by food bacteria such as PF[3] is receiving increasing attention for their potential applications in improving both texture and the health properties of foodstuff. In this work, we explored the genetic and phenotypic bases for the surface properties of the EPS-producing PF strain P52. In this purpose, the whole genomes of the EPS-producing PF P52 strain and of its spontaneous mutant, with an altered EPS-phenotype, were analyzed to identify genetic determinants for EPS biosynthesis. Using lectin-grafted AFM probes, single-molecule force spectroscopy was used to stretch EPS polymers over immobilized individual bacteria[4,5] of both strains. Results evidenced that spontaneous mutation could lead to 10-fold shortening of the EPS length. Application of the extended Free Join Chain model indicated up to 30×103 Kuhn segments for the EPS-producing mutant, vs 5×103 for the mutant. Figure 1: Typical deflection images and single-molecule force curves obtained by AFM analysis of the wild-type EPS-producing P. freudenreichii strain (left) and of its spontaneous mutant (right) Combination of genetic data and force spectroscopy observations provided insight on the biosynthesis of EPS with variable lengths, therefore on the control of thickness of the bacterial capsule for specific food applications.

Food fermentation and biopreservation processes involve the use of various species and strains of bacteria and yeast. These strains are responsible for the targeted qualities of the food products that are sanitary, organoleptic (aroma and texture) and healthy qualities. The Florilege database project aims at (i) gathering bacterial and yeast phenotypes of food product of interest that are automatically extracted from PubMed-referenced full-text litterature by using a text mining approach (ii) managing the information in a relational database (iii) enabling multi-criteria requests via a Web user-friendly interface. To date 368 phenotypes, 260 synthetised or degraded molecules, 1076 medium or food products, 1181 bacterial taxons have been acquired by a combinaison of automatic and manual annotations of text, used for training the text-mining method.Food products are automatically categorized in Florilege according to the OntoBiotope ontology that we have extended with dairy and bakery products definitions. Taxa are categorized by the NCBI taxonomy. An ontology of microbial characteristics has been specifically enriched by the Florilege project. This ontology defines microbial phenotypes (Ontobiotope-Phenotype), including intracellular characteristics of cells (such as shape, antibiotic resistance...) and microbial uses (Ontobiotope-Use) that express the microbial alteration of the external environment, food or matrix, such as aroma, vitamin or other molecule production, degradation or food coloring.A preliminary Web interface is available for querying taxa, culture medium and food products at http://genome.jouy.inra.fr/Florilege/. The public availability of Florilege database is planned for the end of 2017 with a user-friendly interface for multi-criteria requests and access to various phenotypes.Florilege will be a highly valuable tool to (i) assess phenotypic biodiversity of food microbes (ii) assign biochemical functions to each strain/species from fermented or biopreserved food products (iii) help into the development of innovative food products in particular those that involve fermentation or biopreservation processes.

International audience; Les aliments fermentés traditionnels sont essentiels dans les régimes alimentaires méditerranéens et constituent également un héritage culturel et gastronomique riche. La fabrication et les saveurs associées à ces aliments reposent sur l’activité des flores microbiennes naturellement présentes dans l’environnement de production. Ces consortia microbiens parfois très complexes sont pourtant peu caractérisés, malgré leur contribution majeure à la typicité mais également à la durée de vie et aux qualités sanitaires des produits fermentés. En conséquence, une amélioration du management des ressources microbiennes serait nécessaire pour aider les producteurs locaux à améliorer la qualité sanitaire de leur production et in fine améliorer leur durée de vie tout en préservant leur authenticité.Dans le cadre du projet européen (Arimnet2) « PROMEDFOODS » coordonné par le Professeur Jérôme Mounier, un travail de thèse sera initié en étroite collaboration avec des producteurs locaux autour de deux aliments fermentés traditionnels : les olives noires de table et un fromage au lait cru. Les objectifs pour le LUBEM-INRA seront (i) d’étudier la diversité microbienne (bactérienne et fongique) associée à ces deux produits (ii) de suivre leur dynamique au cours de la fabrication via des approches culture-dépendantes (i.e. préservation des ressources microbiennes) et -indépendantes (i.e. métabarcoding et métagénomique), (iii) d’identifier et sélectionner des souches dominantes bénéfiques et (iv) de mettre à profit ces souches chez le producteur pour améliorer le contrôle de la fermentation et la qualité des produits. L’ensemble de ces résultats contribuera à une connaissance approfondie de la diversité taxonomique et fonctionnelle des microorganismes autochtones des aliments fermentés traditionnels.

International audience; Surfactants are compounds with hydrophobic and hydrophilic moieties. Due to this dual nature, surfactants have the ability to partition into the oil-air or oil-water interfaces, to reduce the interfacial and surface tensions. Surfactants are mainly of chemical origin and have broad spectrum of applications such as cleaning agents, environmental bioremediation, pharmaceutics. A large variety of micro-organisms produce surfactants BSF, called biosurfactants (BSF), which vary in their chemical properties and molecular size, and with properties such as low toxicity, high biodegradability. Today, little is known about the BSF production by food bacteria as dairy propionibacteria, that are widely used for cheese making and probiotic applications.In this study we tested 75 strains belonging to the species Propionibacterium acidipropionici, freudenreichii, jensenii, and thoenii for the production of BSF. Production was evaluated by the « drop collapse method », which estimates the surface tension. The whole cultures, the supernatants and the bacterial pellets were tested in order to access the localisation of the BSF. For the positive strains, the surface tension of the BSF production was determined using a tensiometer. The results led to the identification of 21 strains producing BSF. According to the strain, the surface tension of the can reach 52.1 mN/m (when surface tension of egg-lecithin is about 54 mN/m, and the surface tension of water is about 72 mN/m). This works represents the first screening of dairy propionibacteria for the production of BSF and allows to consider bacterial BSF as an alternative to chemically synthesized surfactants.

Propionibacterium freudenreichii is a beneficial bacterium used as vitamin producer, bio-preservative, cheese ripening starter and probiotic. It adheres to intestinal epithelial cells and mucus and modulates the immune response. Interaction with the host is strain-dependent and relies on key surface proteins, still poorly investigated. We identified surface-exposed proteins in a strain previously selected for its probiotic properties. The role of these proteins in immunomodulatory properties was evidenced. Cheese being a major source of bacteria, we developed an experimental pressed cheese, exclusively fermented by a single strain of P. freudenreichii. Key surface proteins involved in the observed immunomodulatory effects were expressed within the cheese matrix. Consumption of this single-strain cheese protected mice from acute colitis induced by TNBS. P. freudenreichii expressed the key genes of stress tolerance response during growth in the aqueous phase of Emmental cheese, in agreement with enhanced stress tolerance, compared to laboratory culture medium.This work confirms the probiotic potential of P. freudenreichii, it provides a new functional fermented product for preclinical and clinical studies aimed at prevention or treatment of IBD. It constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context.

Propionibacterium freudenreichii is a food grade bacterium consumed both in cheeses and in probiotic preparations. Its promising immunomodulatory potential largely relies on the presence of specific and strain-dependent surface compounds. The CIRM BIA 129 strain was selected for immunomodulatory properties, its surfaceome deciphered using an integrative approach. Surface Layer Associated Proteins (SLAPs) are involved in the anti-inflammatory properties

International audience; Paracrystalline bacterial surface protein layers (S layers) are common constituents of the bacterial cell wall. These layers originate from the assembly of strain-variable single protein species, the S layer proteins (SLPs), into paracrystalline lattices that eventually cover the entire surface of the bacteria [1]. S layers are thought to play a role in maintaining integrity of the bacteria or in mediating cell-host crosstalk through surface recognition [2]. Thus, S layer-carrying bacteria can show interesting probiotic potential [3]. Such functions may depend on the actual structure of both the SLP and their paracristalline assembly when delivered into the host’s intestine, i.e., after food preparation and ingestion. When isolated, the SLPs spontaneously self-assemble into mono or bilayer paracrystalline arrays on a wide range of substrates and can be used as models for the detailed investigation of bacterial S layers. In the probiotic Propionibacterium freudenreichii, SLPs were shown to play a strain-dependent central role in bacterium/host interactions, including immunomodulation [4]. In the present study, SLP A, isolated from P. freudenreichii strain CIRM BIA 118 was recrystallized at 25°C and pH 6.7 in HEPES buffer then submitted to either increasing temperatures up to 45°C or decreasing pH values down to pH 3.0, relevant to foodmaking and digestion. In the initial conditions, SLP A assembled in hexagonal paracrystalline bilayer as evidenced by atomic force microscopy (AFM). Solid-state Nuclear Magnetic Resonance (NMR) indicated that the internal structure of this protein, (in the S layer array or in solution) exhibited many (~70%) disordered regions, also containing significant amount of bound water. When submitted to heating at 35 or 45°C, the structure of the paracrystalline array was maintained but exhibited decreasing elasticity as probed by atomic force spectroscopy (AFS). When submitted to acidification, the structure and elasticity of the paracrystalline array was maintained at pH 5.0 but varied at pH 3.0. The results were interpreted in terms of changes in the exposed disordered regions of the protein when assembled in the S layer. Such changes may determine the nature and extent of bacterium/host interactions.The authors aknowledge Valérie Briard for mass spectrometry identification of SLP A.

Propionibacterium freudenreichii is the main Propionibacterium consumed both in traditional cheeses and in innovative probiotic product. A portion of 10 grams of Emmental cheese provides around 10 10 live P. freudenreichii , a major bacterial intake in France and other industrialized countries. Surprisingly, little is known about its effect on human health, compared to lactobacilli or bifidobacteria. However, recent works indicate promising beneficial effects including modulation of intestinal microbiota, metabolic activities and inflammation. In this context, the molecular mechanisms responsible for the immunomodulatory properties of P.freudenreichii , and of fermented dairy product containing it, were investigated. In that aim, an integrative approach was undertaken, in a research program of the French National Agency for Research (ANR). It consisted of the sequencing and annotation of the genomes of 20 strains, followed by comparative genomics, in the design of a pangenomic chip, followed by comparative transcriptomic analysis , in a comparative surface proteomic analysis, and in comparison of the immunomodulatory properties, ex vivo and in vivo , using model cheeses. This investigation reveals, for the first time, the molecular mechanism responsible for modulation of the immune system and of inflammation by P.freudenreichii . These results are in accordance with clinical results obtained by others with the same bacterium

This book underlines the importance of reciprocal interactions between probiotics and humans in terms of stress induction, epigenetic control of cellular responses, oxidative status, bio-active molecules biosynthesis, moonlighting proteins secretion, endogenous toxins neutralization, and several other biological functions. It explores how these responses can affect metabolism and metabolic-related disorders, gutbrain axis balance, mood, inflammatory, allergic and anti-infective reactions, cancer, and ageing. The book explores how probiotics create a dynamic and "fluid" network of signals able to control the balance between healthy and altered human status.

Propionibacterium freudenreichii is consumed in elevated amounts both as a cheese starter (1010 CFU/D/person in France) and as a probiotic bacterium, while its effect on health is poorly understood. We investigate its immunomodulatory properties, in a context of growing occurrence of inflammatory bowel diseases. Twenty strains were compared in this regard in vitro using human PBMCs (Peripheral Blood Mononuclear cells), revealing strain dependent cytokine stimulatory properties. They also afforded strain-dependent protection in vivo towards colitis. A fermented milk, exclusively fermented by P. freudenreichii, was developed and tested in a healthy piglet model. It promoted growth health of the piglets and delivered live and metabolically active propionibacteria to the gut. Its consumption reduced gut mucosa production of pro-inflammatory IL-8 and TNF-α. Among strains, a genomic, transcriptomic and proteomic approach revealed contrasted effects of propionibacterial surface compounds such as exopolysaccharides and proteins. Specific proteins are involved in immunomodulation, while capsular exopolysaccharides abolish it. The role of each key compound is presently investigated in an ANR project (Sarter SURFace against INflammation of the Gut) in a multidisciplinary approach involving 7 partners. Elucidation of molecular and cellular mechanisms, together with in vivo anti-inflammatory effects, will open new perspectives for the use of fermented dairy products containing P. freudenreichii addressing the needs of specific populations.

L'Unité de Recherche Œnologie (EA4577 de l’Université Bordeaux Segalen et USC1366 INRA, rattachée au département CEPIA - Caractérisation et Elaboration des Produits issus de l'Agriculture), organise la 19ème édition du Club des Bactéries Lactiques.; Dairy propionibacteria (PAB) are used as a ripening starter in combination with Lactic acid bacteria (LAB)for dairy products such as Swiss-type cheese. LAB and PAB have also been studied for their probioticproperties but little is still known about their individual and/or synergistic beneficial effects within dairymatrices. In the context of a rising incidence of Inflammatory Bowel Diseases, it has become crucial toevaluate the immunomodulatory potential of bacteria ingested in large numbers via dairy products. Wetherefore selected different strains and combinations of technological LAB and PAB. We determined theirimmunomodulatory potential by IL-10 and IL-12 induction, in human peripheral blood mononuclearcells, on either single or mixed cultures, grown on laboratory medium or directly in milk. Milk wasfermented with selected anti-inflammatory strains of LAB or PAB/LAB mixed cultures and the resultingbacterial fractions were also evaluated for these properties, together with starter viability and optimumtechnological aspects. The most promising fermented milks were evaluated in the context of TNBS- orDSS-induced colitis in mice. The improvement in inflammatory parameters evidenced an alleviation ofcolitis symptoms as a result of fermented milk consumption. This effect was clearly strain-dependent andmodulated by growth within a fermented dairy product. These findings offer new tools and perspectivesfor the development of immunomodulatory fermented dairy products for targeted populations.

Surface proteins are key actors of the complex interactions between bacteria (pathogens, commensals or symbionts) and their host. In beneficial (probiotic) bacteria, they participate in competition with pathogens, adhesion to the host cells, and immunomodulation. We investigated such proteins in the beneficial bacterium Propionibacterium freudenreichii, consumed both in Swiss-type cheeses and probiotic preparations. P. freudenreichii genome was sequenced and annotated, the localization of the encoded proteins was predicted using SurfG+. A combination of 3 biochemical methods confirmed surface exposure of P. freudenreichii proteins: shedding, shaving and labelling. Shedding consisted in the extraction of cell-wall associated proteins using guanidine, followed by trypsinolysis of the extracted proteins. Shaving consisted in enzymatic hydrolysis of surface protruding proteins which were accessible to trypsin in situ on live bacteria. For labeling, an NHS-ester-cyanine was added to live bacteria in order to label surface proteins, prior to 2-D electrophoresis and detection of fluorescent protein spots. For the 3 methods, the resulting tryptic peptides were identified by NanoLC-MS/MS on a Q-TOF mass spectrometer This combination of methods allowed identification of surface layer type-proteins, lipoproteins, proteins associated to the cell wall, to the membrane, or predicted to be secreted, as well as moonlighting proteins predicted to be cytoplasmic. Some of these proteins are known to participate in adhesion and in the modulation of the immune response by probiotics. This work constitutes a decisive step in the elucidation of P. freudenreichii ability to interact with host cells and in the understanding of protein sorting in this bacterium.

Dairy propionibacteria, mainly Propionibacterium freudenreichii, are consumed in high amounts within cheeses. They are also known as probiotics able to modulate the gut microbiota and the corresponding metabolic activities. Their immunomodulatory properties, analyzed here on human PBMCs, displayed a remarkable variability, depending on the P. freudenreichii strain. Moreover, such modulating properties varied depending on the culture medium considered, i.e. laboratory culture medium versus fermented dairy product. Selected anti-inflammatory strains were further tested in 2 distinct mice experimental colitis models, revealing significant protection and confirming for these cheese starter bacteria (i) probiotic potential and (ii) predictive value of immune-based selection criteria. The role of surface component, including surface exopolysaccharide (EPS) and surface exposed proteins (SEPs), in this modulation, was investigated. We showed that presence of EPS abrogates immunomodulation while presence of key SEPs determines nature and intensity of this modulation. These key SEPs are being identified, as well as the mechanisms leading to their variability. These results open new perspectives for propionibacteria selection aiming at probiotic applications and new mechanistic explanation of their effects on the digestive tract.

Propionibacterium freudenreichii est une bactérie de l’ordre des Actinomycetales qui bénéficie des statuts GRAS et QPS. Consommée en grande quantité (1010 CFU/jour/personne en France) dans les fromages de type Emmental, elle est également commercialisée en tant que probiotique. Cette étude vise à mettre en évidence les propriétés immunomodulatrices d’une telle consommation, dans un contexte où les maladies intestinales inflammatoires constituent un problème de santé publique croissant. Sujets / matériels et méthodes : Les propriétés immunomodulatrices de 20 souches de P. freudenreichii ont été comparées in vitro dans un modèle de PBMC humains, puis in vivo dans deux modèles de colite expérimentale chez la souris. Un lait fermenté exclusivement par P. freudenreichii a été mis au point et testé dans un modèle de porcelet sain. Une approche multidisciplinaire (purification, mutation, expression hétérologue, évaluation in vitro et in vivo) précise les mécanismes mis en jeux. Résultats principaux : Nous avons mis en évidence des propriétés immunomodulatrices souche-dépendantes pour cette bactérie, en terme de stimulation de cytokines et de protection vis-à-vis de colite expérimentale chez la souris. Le lait fermenté mis au point a eu un effet promoteur de santé chez le porcelet et délivre P. freudenreichii vivant et métaboliquement active dans le côlon. Sa consommation réprime la production de la cytokine pro-inflammatoire IL-8 au niveau de la muqueuse intestinale porcine. Une approche mécanistique a révélé les rôles contrastés de différents composés de surface tels que les exopolysaccharides et les protéines de surface, dans l’immunomodulation. Certaines protéines sont impliquées dans la modulation du système immunitaire, alors que la capsule exopolysaccharidique annule cette modulation. Conclusions : Le rôle de chaque molécule est actuellement abordé dans un projet ANR (Sarter SURFace against INflammation of the Gut) par une approche multidisciplinaire mobilisant 7 partenaires. L’élucidation des mécanismes cellulaires et moléculaires induits, ainsi que les effets observés in vivo, ouvriront des perspectives pour l’utilisation de produits laitiers fermentés par P. freudenreichii répondant aux besoins spécifiques de populations ciblées.

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