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In this study, the potential for a collection of wild-type L. lactis strains to metabolize the breakdown products of lignocellulose was investigated. The strains, isolated from a variety of environmental sources including grass and vegetables, were analyzed for their ability to ferment pentose sugars and their cellulolytic ability. In total, 21 environment-derived L. lactis strains were evaluated. Eleven of the 21 L. lactis isolates were found to have the potential to ferment pentose sugars commonly produced by lignocellulose breakdown. A 3,5-dinitrosalicylic acid (DNS)-based cellulase assay was performed, and 10 of the 21 L. lactis isolates showed cellulolytic activity. Six strains were able to both metabolize pentose sugars and showed cellulolytic activity: these included green pea isolates DPC 6754, DPC 6755, DPC 6756, and DPC 6758, the grass isolate DPC 6760, and the mung bean sprouts isolate KF147. For the first time, certain wild-type non-engineered L. lactis were found to possess cellulolytic activity. Moreover, these two abilities do not appear to be correlated. These findings highlight that environment-derived L. lactis, a species with a history of safe use in food production, has the potential for second-generation bioconversion processes, and the potential to re-utilize plant biomass found in waste streams.

International audience; 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.

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.

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 cocultures 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 cocultures, 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 nonproteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was cocultured with each one of the nonproteolytic LAB strains, L. lactis NCDO2111 and Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and cocultures. Acidification, carbohydrate consumption, and volatile-compound production were evaluated in direct cocultures. Each proteolytic strain induced different types of interactions: strongly positive interactions, weakly positive interactions, and no interactions were seen with E. faecalis CIRM-BIA2412, L. lactis NCDO2125, and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations of tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to higher acidification rates, lower pH, higher raffinose utilization, and higher concentrations of five volatile compounds. IMPORTANCE Interactions of lactic acid bacteria (LAB) 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 investigated the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and nonproteolytic strains. The results suggest that proteolytic LAB do not equally stimulate nonproteolytic 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 guarantee their positive interactions.

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

International audience; Background: Since the early 1990s, major health and environmental concerns have developed and driven the emergence of diets involving a lower consumption of animal products. However, the transition towards greener diets is being hampered by the poor acceptance of vegan foodstuffs among western consumers. Mixed animal/ plant alternatives to familiar dairy or egg products offer a new field of innovation. Scope and approach: This review focuses on innovative mixes of egg or milk with plant ingredientsespecially legumesto develop products in which interactions between animal and plant are not usually expected, such as dairy or egg gels, emulsions or foams. The opportunities offered by such products in terms of consumer acceptance, nutrition, digestibility and techno-functional properties are reviewed and discussed with respect to their risk-benefit ratios. Key findings and conclusions: In many cases, animal/plant mixes offer enhanced protein stability and synergistic interfacial or textural properties that make them a flexible tool for food design. Fermentation offers important prospects for the nutritional and sensorial enhancement of animal/plant mixes, through the multi-criteria application of microbial consortia. Animal/plant mixes enable reduction in animal protein consumption while preserving amino acid and micronutrient intakes and sensory properties. However, their acceptability to consumers and society will also depend on controlled safety, especially regarding allergies or contaminants, on affordability, their degree of novelty or (ultra)processing, their actual environmental footprint and whether they meet consumer expectations for innovative foods in the transition towards greener diets.

Grâce aux travaux de son équipe en « expérience patient », le centre hospitalier universitaire (CHU) de Québec-Université de Laval a inscrit comme prioritaire la prise en compte de l’expérience des patients et de leurs proches dans ses orientations stratégiques dès 2014. Dans le cadre d’un appel à projets de coopération internationale hospitalière, le CHU de Rouen s’est inspiré de cette démarche. Des échanges entre des professionnels des deux établissements ont permis d’orienter la méthodologie de co-construction du nouveau projet stratégique 2020-2024 du CHU de Rouen ainsi que ses objectifs, en mettant au centre de cette nouvelle vision l’expérience des patients et le partenariat avec ceux-ci. L’objectif poursuivi par les deux CHU est de définir une vision claire, porteuse de sens, qui permette aux professionnels de s’impliquer conformément aux grands principes de management de la qualité reconnus au niveau international.

The famous French dessert "ile flottante" consists of a sweet egg white foam floating on a vanilla custard cream, which contains highly nutritive raw materials, including milk, sugar and egg. Spoilage issues are therefore a key concern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream desserts manufactured by 2 French companies. B. cereus group bacteria, coagulase negative Staphylococcus, Enterococcus and Leuconostoc spp. were isolated from spoiled products. Thirty-one bacterial isolates representative of the main spoilage species were tested for their spoilage abilities. Significant growth and pH decrease were observed regardless of species. While off-odours were detected with B. cereus group and staphylococci, yoghurt odours were detected with Enterococcus spp. and Leuconostoc spp. B. cereus group bacteria produced various esters and several compounds derived from amino acid and sugar metabolism. Most Staphylococci produced phenolic compounds. Enterococcus spp. and Leuconostoc spp. isolates produced high levels of compounds derived from sugar metabolism. Each type of spoilage bacteria was associated with a specific volatile profile and lactic acid was identified as a potential marker of spoilage of custard cream-based desserts. These findings provide valuable information for manufacturers to improve food spoilage detection and prevention of chilled desserts made with milk and egg.

Domestication provides an excellent framework for studying adaptive divergence. Using population genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese mould Penicillium roqueforti. We showed that this fungus was domesticated twice independently. The population used in Roquefort originated from an old domestication event associated with weak bottlenecks and exhibited traits beneficial for pre‐industrial cheese production (slower growth in cheese and greater spore production on bread, the traditional multiplication medium). The other cheese population originated more recently from the selection of a single clonal lineage, was associated with all types of blue cheese worldwide except Roquefort, and displayed phenotypes more suited for industrial cheese production (high lipolytic activity, efficient cheese cavity colonization ability and salt tolerance). We detected genomic regions affected by recent positive selection and putative horizontal gene transfers. This study sheds light on the processes of rapid adaptation and raises questions about genetic resource conservation., see also the Perspective by Brigida Gallone, Jan Steensels and Kevin J. Verstrepen.

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; 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.

An egg product is a complex matrix, mainly composed of water, proteins and lipids. It also contains carbohydrates, vitamins, minerals and many other compounds in varying concentrations. This chemical complexity makes it difficult to analyze, and most of the protocols are mainly concerned with the albumen and vitellus fractions, aside from the whole egg product. The hygienic quality and freshness of eggs can be assessed according to physico-chemical or microbiological criteria, imposed by the regulations in force. The state provides procedures and recommendations for the production and marketing of such products. Criteria and procedures vary from region to region, for example European legislation forbids the breaking of eggs by centrifugation for eggs intended for the food industry whereas American legislation allows the possibility of breaking eggs by centrifugation if they are clean, disinfected and intact and if they ultimately undergo heat treatment (baking etc.). In addition to the procedures and recommendations concerning the production process, the hygienic quality of egg products is ensured by controlling the physico-chemical and microbiological quality of the raw materials used and the finished products. European legislation thus provides maximum levels for certain compounds such as lactic acid or succinic acid at different points in the production chain.

Biopreservation represents a complementary approach to traditional hurdle technologies for reducing microbial contaminants (pathogens and spoilers) in food. In the dairy industry that is concerned by fungal spoilage, biopreservation can also be an alternative to preservatives currently used (e.g. natamycin, potassium sorbate). The aim of this study was to develop antifungal fermentates derived from two dairy substrates using a sequential approach including an in vitro screening followed by an in situ validation. The in vitro screening of the antifungal activity of fermentates derivating from 430 lactic acid bacteria (LAB) (23 species), 70 propionibacteria (4 species) and 198 fungi (87 species) was performed against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum and Yarrowia lipolytica) using a cheese-mimicking model. The most active fermentates were obtained from Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei/paracasei and Lactobacillus plantarum among the tested LAB, Propionibacterium jensenii among propionibacteria, and Mucor lanceolatus among the tested fungi. Then, for the 11 most active fermentates, culture conditions were optimized by varying incubation time and temperature in order to enhance their antifungal activity. Finally, the antifungal activity of 3 fermentates of interest obtained from Lactobacillus rhamnosus CIRM-BIA1952, P. jensenii CIRM-BIA1774 and M. lanceolatus UBOCC-A-109193 were evaluated in real dairy products (sour cream and semi-hard cheese) at a pilot-scale using challenge and durability tests. In parallel, the impact of these ingredients on organoleptic properties of the obtained products was also assessed. In semi-hard cheese, application of the selected fermentates on the cheese surface delayed the growth of spoilage molds for up to 21 days, without any effect on organoleptic properties, P. jensenii CIRM-BIA1774 fermentate being the most active. In sour cream, incorporation of the latter fermentate at 2 or 5% yielded a high antifungal activity but was detrimental to the product organoleptic properties. Determination of the concentration limit, compatible with product acceptability, showed that incorporation of this fermentate at 0.4% prevented growth of fungal contaminants in durability tests but had a more limited effect against M. racemosus and P. commune in challenge tests. To our knowledge, this is the first time that the workflow followed in this study, from in vitro screening using dairy matrix to scale-up in cheese and sour cream, is applied for production of natural ingredients relying on a large microbial diversity in terms of species and strains. This approach allowed obtaining several antifungal fermentates which are promising candidates for dairy products biopreservation.

Algerian couscous named lemzeiet is manufactured from fermented wheat. Historically performed in underground silos called matmor, the fermentation of wheat is now generally carried out in plastic jerrycans with or without addition of vinegar at the beginning of the fermentation. Culture-dependent and culture-independent methods (PCR-TTGE) were used to characterize lactic acid bacteria and to determine their dynamic and diversity over a two-year period, with and without the addition of vinegar. Fungi, physicochemical characteristics, and volatile compound profiles were also monitored. The isolates obtained from different stages of fermentation and from both processes were characterized by coupling different molecular methods (16SrRNA/pheS/rpoA gene sequencing, species-specific PCR, RAPD and PFGE). PCR-TTGE revealed very similar profiles for both processes. Sixty-nine isolates were identified as belonging to six genera of 16 species (Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Weissella, and Streptococcus). The profiles of volatile aroma compounds showed a marked effect of the fermentation process, compared to non-fermented wheat, with 35 of the 40 volatiles detected at amounts 20- to 30-fold higher in fermented wheat samples. This study gives the first insight into lactic acid bacteria population diversity and activity in fermented wheat and will contribute to a better control of the fermentation process.

Esters are important flavour-active compounds in cheese and result from the activity of cheese bacteria. The bacteria and reactions involved, however are still largely unknown, with two main mechanisms, alcoholysis or esterification, potentially involved in ester synthesis from different precursors. The aim of this study was to compare the potential for ester synthesis via different reactions in ripening bacteria. Four strains, one Lactobacillus fermentum, one L. casei, and two Propionibacterium freudenreichii, were tested as growing cultures and cell-free extracts (CFE) for their capacity to synthesise ethyl butanoate (EtC4) in the presence of different precursors. In cultures, EtC4 was mainly produced via alcoholysis. The highest EtC4 levels were produced by L. fermentum. Propionibacteria were also able to synthesise esters via alcoholysis, as demonstrated here for the first time. CFE from L. fermentum synthesised EtC4 via alcoholysis, whereas the other three strains did it via esterification. Our results show that the EtC4 synthesis mechanisms and the amounts produced were markedly strain-dependent, and suggest that both esterification and alcoholysis could be involved in ester formation during cheese ripening. By providing a better understanding of EtC4 biosynthesis, this work paves the way for controlling its production in cheese, accelerating the formation of desirable flavour. Fil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Maillard, Marie Bernadette. Institut National de la Recherche Agronomique; Francia Fil: Medina, Roxana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina. Universidad Nacional de Tucumán; Argentina Fil: Thierry, A.. Institut National de la Recherche Agronomique; Francia

Enzymatic hydrolysis (lipolysis) of milk glycerides to free fatty acids is essential for flavor development in cheese. The principal lipolytic agents in cheese include lipoprotein lipase from raw milk, pregastric esterase in cheeses made using rennet paste, and enzymes from the starter and nonstarter microbiota. Lactic acid bacteria are weakly lipolytic and mainly possess nonlipolytic esterases located intracellularly. Lipolysis level is, thus, low in many internal bacterially ripened cheeses. It is higher in certain varieties, such as Swiss cheese, smear-ripened, and particularly mold-ripened cheeses, in which specific lipolytic secondary microbiota develops. Exogenous lipases are occasionally used to develop flavor. Short-chain fatty acids directly contribute to flavor, but fatty acids can also act as precursors for the production of a wide range of other flavor compounds, such as esters, lactones, and methylketones that are associated with diverse flavors. Methods for determining levels of fatty acids are also discussed. Fil: Thierry, Anne. Institut National de la Recherche Agronomique; Francia Fil: Collins, Yvonne F.. Teagasc Food Research Centre; Irlanda Fil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad del Norte Santo Tomás de Aquino; Argentina Fil: McSweeney, Paul LH.. University College. School of Food and Nutritional Sciences; Irlanda Fil: Wilkinson, Martin G.. University of Limerick; Irlanda Fil: Spinnler, Henri E.. Institut National de la Recherche Agronomique; Francia

peer-reviewed Cheese flavor development is directly connected to the metabolic activity of microorganisms used during its manufacture, and the selection of metabolically diverse strains represents a potential tool for the production of cheese with novel and distinct flavor characteristics. Strains of Lactobacillus have been proven to promote the development of important cheese flavor compounds. As cheese production and ripening are long-lasting and expensive, model systems have been developed with the purpose of rapidly screening lactic acid bacteria for their flavor potential. The biodiversity of 10 strains of the Lactobacillus casei group was evaluated in 2 model systems and their volatile profiles were determined by gas chromatography-mass spectrometry. In model system 1, which represented a mixture of free AA, inoculated cells did not grow. In total, 66 compounds considered as flavor contributors were successfully identified, most of which were aldehydes, acids, and alcohols produced via AA metabolism by selected strains. Three strains (DPC2071, DPC3990, and DPC4206) had the most diverse metabolic capacities in model system 1. In model system 2, which was based on processed cheese curd, inoculated cells increased in numbers over incubation time. A total of 47 compounds were identified, and they originated not only from proteolysis, but also from glycolytic and lipolytic processes. Tested strains produced ketones, acids, and esters. Although strains produced different abundances of volatiles, diversity was less evident in model system 2, and only one strain (DPC4206) was distinguished from the others. Strains identified as the most dissimilar in both of the model systems could be more useful for cheese flavor diversification.

1- Introduction: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. 2- Objectives : The aim of the study was to decipher metabolic adaptations of P. freudenrechii responsible for long-term survival under gradual nutritional shortage. 3- Materials & methods: For 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. The strain with the highest survival rate was incubated for 11 days (9 days after entry into stationary phase) in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, without any adjunct during the incubation. Its physiological adaptation was investigated by RNA-seq analysis. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. 4- Results: Under these conditions, the eight studied strains displayed high survival rates, their culturability reaching more than 9 log 10 CFU/ml after 2 days. After 11 days, this value ranged from 7.8 to 8.2 log 10 CFU/ml depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. P. freudenreichii with the highest survival rate was CIRM-BIA 138 (alias ITG P9). Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the CIRM-BIA 138 strain sustained a high population level of 9.3 log 10 CFU/mL and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. 5- Conclusions: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle, by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

New strains are desirable to diversify flavour of fermented dairy products. The objective of this study was to evaluate the potential of Leuconostoc spp. and Lactobacillus spp. in the production of aroma compounds by metabolic fingerprints of volatiles. Eighteen strains, including five Lactobacillus species (Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus sakei) and three Leuconostoc species (Leuconostoc citreum, Leuconostoc lactis, and Leuconostoc mesenteroides) were incubated for 5 weeks in a curd-based slurry medium under conditions mimicking cheese ripening. Populations were enumerated and volatile compounds were analysed by headspace trap gas chromatography– mass spectrometry (GC–MS). A metabolomics approach followed by multivariate statistical analysis was applied for data processing and analysis. In total, 12 alcohols, 10 aldehydes, 7 esters, 11 ketones, 5 acids and 2 sulphur compounds were identified. Very large differences in concentration of volatile compounds between the highest producing strains and the control medium were observed in particular for diacetyl, 2-butanol, ethyl acetate, 3- methylbutanol, 3-methylbutanoic acid and 2- methylbutanoic acid. Some of the characterized strains demonstrated an interesting aromatizing potential to be used as adjunct culture.

In cheese technology, the diffusion phenomena are crucial during ripening. The technique of Fluorescence Recovery After Photobleaching was applied for the first time on real cheese, in order to investigate the relationships between molecular diffusion and the cheese composition and/or its microstructure. Measured effective diffusion coefficients in soft and hard cheese of a group of dextrans (10-500 kDa) were found to be in the same order of magnitude with values observed when using a comparable non-fat model cheese (∼ 0.1-20 μm(2) s(-1)). Diffusion of the dextrans was mainly dependent on the fraction of "free" aqueous phase present in the cheese, closely which is linked to cheese-making technology and ripening stage. Diffusion coefficients were modeled by a power law relationship as a function of dextran molecular weight, which allowed some study of the cheese microstructure. A tighter protein network will require some deformation of those flexible macromolecules with a higher molecular weight (>250 kDa), in order to diffuse through the pores of such cheese structures.

– Cantal is the third French Protected Denomination of Origin cheese. The Cantal cheesemaking process is similar to Cheddar cheese. Both processes involve a milling and a dry salting step which results in a homogeneous salted curd. In order to determine the key parameters of the manufacturing process acting on the specific characteristics of this cheese variety, an extensive and kinetic study repeated three times was carried out at nine stages from the raw milk to 120 d ripened cheese. The microbial ecosystem was explored by numeration and by direct extraction of DNA and PCR-Temporal Temperature Gradient Electrophoresis. Lysis of the lactic acid bacteria was checked by quantification of the intracellular lactate dehydrogenase activity. Carbon sources changes, solubilisation of minerals, proteolysis, lipolysis, and formation of neutral volatile compounds were also assessed. Cheese microstructure modifications and bacterial localisation were determined by confocal scanning laser microscopy. The controlled manufacturing conditions and the use of a complex mixture of starters led to very similar ripened cheeses. The compositional attributes (in particular total solids, fat, nitrogen forms), the lipolysis (0.7 ± 0.2% fat) and proteolysis final extent (34.4 g·kg–1 free amino acids in juice) were similar as well as major neutral volatiles compounds (2-butanone, ethanol and 2-butanol). Nevertheless, lactose and citrate metabolisms differed between the three trials and some free amino acids (Arg, Pro, Ser, Cit, Homocys) concentrations varied drastically during the ripening time course. CLSM micrographs showed that fat globules were disrupted during pressing and bacteria were preferentially localised at the fat/protein interface. As the lactic starter species were the same in the three trials and predominant during the cheese manufacture, the observed differences could result of one or several of the followings: (i) the raw milk microflora which showed to be different leading to growth or metabolism activity of particular subdominant populations during the process and the ripening; (ii) the more or less early lysis of the lactic starters; (iii) the possible interactions between raw milk microflora and the added starters. This extensive characterization of Cantal cheese provides a strong basis for building a rational concept of the key steps in which either technological actions or complementary microbial inoculation could be done in order to limit day to day variations.; Le fromage de Cantal est, en tonnage, la 3ème Appellation d'Origine Contrôlée française. Son procédé de fabrication, similaire à celui du Cheddar, implique une étape de broyage et salage à sec, qui résulte en un caillé salé homogène. Afin de déterminer les paramètres clé du procédé de fabrication agissant sur les caractéristiques spécifiques de cette variété de fromage, une étude approfondie et cinétique avec trois répétitions a été menée à 9 stades, du lait cru au fromage affiné 120 jours. L'écosystème microbien a été exploré par énumération et par extraction directe de l'ADN et PCR-Temporal Temperature Gradient Electrophoresis. La lyse des bactéries lactiques a été appréhendée par mesure de l'activité de la lactate déshydrogénase intracellulaire. Les changements intervenant dans les sources carbonées, la solubilisation des minéraux, la protéolyse, la lipolyse et l'apparition des composés volatiles neutres ont aussi été évalués. Les modifications de la microstructure du fromage ainsi que la localisation des bactéries ont été déterminées pas microscopie confocale à balayage laser (CLSM). La mise en oeuvre de conditions de fabrication très contrôlées et l'utilisation d'un levain complexe a conduit à des fromages affinés très similaires, tant sur le plan de la composition (en particulier l'extrait sec total, la matière grasse, les formes azotées) des niveaux de lipolyse (0.7 ± 0.2 % de la matière grasse) et de protéolyse (34,4 g·kg-1 d'acides aminés libres dans le jus) que sur le plan des teneurs en composés volatiles majoritaires (2-butanone, éthanol et 2-butanol). Néanmoins, les métabolismes du lactose et du citrate différaient entre les trois essais, ainsi que les concentrations en certains acides aminés libres (Arg, Pro, Ser, Cit, Homocys) qui variaient fortement au cours de l'affinage. Les photographies obtenues par CLSM montraient que les globules gras étaient brisés lors du pressage et que les bactéries étaient préférentiellement localisées à l'interface matière grasse/protéine. Comme le levain utilisé comportaient les mêmes espèces pour les trois essais et que celles-ci s'avéraient dominantes au cours de la fabrication, les différences observées peuvent résulter : (i) des variations observées dans la flore indigène du lait cru conduisant à la croissance ou l'activité métabolique de populations sous-dominantes particulières pendant la fabrication ou l'affinage ; (ii) de la lyse plus ou moins précoce des espèces du levain ; (iii) des interactions possibles entre la flore indigène du lait cru et le levain ajouté. Cette caractérisation approfondie du fromage Cantal offre une base solide pour construire un concept rationnel des étapes-clé au cours desquelles soit des actions technologiques, soit une inoculation microbienne complémentaire peuvent être réalisées afin de limiter les variations journalières et d'optimiser la qualité globale.

Bacteria, either indigenous or added, are immobilized in solid foods where they grow as colonies. Since the 80's, relatively few research groups have explored the implications of bacteria growing as colonies and mostly focused on pathogens in large colonies on agar/gelatine media. It is only recently that high resolution imaging techniques and biophysical characterization techniques increased the understanding of the growth of bacterial colonies, for different sizes of colonies, at the microscopic level and even down to the molecular level. This review covers the studies on bacterial colony growth in agar or gelatine media mimicking the food environment and in model cheese. The following conclusions have been brought to light. Firstly, under unfavorable conditions, mimicking food conditions, the immobilization of bacteria always constrains their growth in comparison with planktonic growth and increases the sensibility of bacteria to environmental stresses. Secondly, the spatial distribution describes both the distance between colonies and the size of the colonies as a function of the initial level of population. By studying the literature, we concluded that there systematically exists a threshold that distinguishes micro-colonies (radius < 100–200 μm) from macro-colonies (radius >200 μm). Micro-colonies growth resembles planktonic growth and no pH microgradients could be observed. Macro-colonies growth is slower than planktonic growth and pH microgradients could be observed in and around them due to diffusion limitations which occur around, but also inside the macro-colonies. Diffusion limitations of milk proteins have been demonstrated in a model cheese around and in the bacterial colonies. In conclusion, the impact of immobilization is predominant for macro-colonies in comparison with micro-colonies. However, the interaction between the colonies and the food matrix itself remains to be further investigated at the microscopic scale.

In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening.

Dairy propionibacteria, and especially Propionibaclerium freudenreichii subsp. shermanii, are important in the food industry and biotechnology. Only a few investigations have focused on the complex physiology of this remarkable bacterium, while the physiology of dairy lactic acid bacteria has been extensively studied over the past decades. Here we report the progress of our whole genome sequencing project: 93% of the P. shermanii genome was assembled in 426 contigs with low overall redundancy. Our annotation strategy overlaps the sequence finishing step, thus improving it. Annotation of the incomplete genome is performed using ContigBrowser, a bioinformatic tool allowing data management, developed in our laboratory. This resulted in the detection of 2611 putative proteins (data May 2003). Our tool allows an expert annotation by manual verification and curation of functional protein categories after automatic assignment. Our genomic sequence analysis, combined with the already developed physiological, proteomic and metabolomic approaches, will allow researchers to explore the significant potentialities of dairy propionibacteria by providing a comprehensive view of the enzymes and metabolic pathways. This knowledge will allow researchers to explore more effective strategies to enhance the utility of this organism in manufacturing procedures or current industrial processes.

Microorganisms play an important role in the development of cheese flavor. The aim of this study was to develop an approach to facilitate screening of various cheese-related bacteria for their ability to produce aroma compounds. We combined i) curd-based slurry medium incubated under conditions mimicking cheese manufacturing and ripening, ii) powerful method of extraction of volatiles, headspace trap, coupled to gas chromatography-mass spectrometry (HS-trap-GC-MS), and iii) metabolomics-based method of data processing using the XCMS package of R software and multivariate analysis. This approach was applied to eleven species: five lactic acid bacteria (Leuconostoc lactis, Lactobacillus sakei, Lactobacillus paracasei, Lactobacillus fermentum, and Lactobacillus helveticus), four actinobacteria (Brachybacterium articum, Brachybacterium tyrofermentans, Brevibacterium aurantiacum, and Microbacterium gubbeenense), Propionibacterium freudenreichii, and Hafnia alvei. All the strains grew, with maximal populations ranging from 7.4 to 9.2 log (CFU/mL). In total, 52 volatile aroma compounds were identified, of which 49 varied significantly in abundance between bacteria. Principal component analysis of volatile profiles differentiated species by their ability to produce ethyl esters (associated with Brachybacteria), sulfur compounds and branched-chain alcohols (H. alvei), branched-chain acids (H. alvei, P. freudenreichii and L. paracasei), diacetyl and related carbonyl compounds (M. gubbeenense and L. paracasei), among others.

Bacteria are present in all foods, whether they are indigenous or inoculated. They can be beneficial to the quality of foods, responsible for food spoilage, or even pathogens. In solid food products, bacteria are immobilized. They thus grow as colonies entrapped within the food products or on the food surfaces. In both cases, bacteria interact with the solid matrix, sometimes facing difficulties to access the nutrients as nutrients have to diffuse from the matrix to the bacterial colonies. Bacterial development can thus be impaired in solid matrices in comparison to planktonic growth. To control the growth of bacteria in solid foods is then of major importance. In the case of pathogens, it is crucial for safety issues to predict how the bacteria present as initial contaminants will develop. In the case of inoculated bacteria, such as lactic acid bacteria, it is also crucial to control their development because they are responsible for the final quality of the food products. However, studies on the growth of bacteria have been essentially focused on growth in liquid media. Resolute techniques, which include both microcopy approaches and quantitative techniques, have now been developed to observe colonies at the microscopic scale. They allow studying the variation of growth in different contexts either in model growth culture media or in model foods. This Research Topic starts with four review papers. A first comprehensive review about bacterial colonies redraws the history of the studies on colonies, synthetizes the conditions of growth in which growth in colonies differs from planktonic growth, and finally presents concepts of the interaction of bacterial colonies with the food matrix in which they grow, with cheese as an example (Jeanson et al.). The second review demonstrates the importance of modeling the growth of bacterial colonies in solids foods despite a lack of knowledge. The different types of models and their potential consequences on decisions are presented and discussed (Skandamis and Jeanson). The third review shows how non-invasive techniques can be used to observe bacterial growth and also quantify the bacterial metabolism in colonies at the microscopic scale (Lobete et al.). The last review paper shows how microscopy techniques are particularly valuable to increase knowledge about bacterial colonies in model foods and foods. Fluorescent microscopy allows targeting metabolites and understand the physiology of bacteria within colonies (Hickey et al.). The five following papers present original results in the field. Using an automated microscope coupled with fluorescence dyes, it was possible to demonstrate that the exposure to an oxidizing disinfectant led to different morphologies of cells depending on the strains and that dead cells were randomly distributed within the micro-colonies (Zhao et al.). The following study demonstrates the accuracy of a phenotyping technology based on laser-induced speckle scatter patterns in Bacillus colonies. The authors showed that the distribution of speckle size is modified during the growth of colonies (Kim et al.). The three following studies investigate bacterial colonies of lactic acid bacteria in their food environment in model cheeses or investigate the pH in their surrounding micro-environment in Cheddar cheeses. Physiology and growth of Streptococcus thermophilus colonies was compared in liquid milk and renneted milk using microcalorimetry. The results showed that above a threshold size of colonies (low inoculation levels), growth rates and metabolism in renneted milk differed from the one observed in liquid milk (Stulova et al.). The use of fluorescence lifetime imaging was shown to be relevant to investigate the pH micro-heterogeneity in Cheddar cheeses (Burdikova et al.). These results suggest that the surroundings of bacterial colonies within cheeses could differ from homogeneous growth conditions in liquid medium. The last study questioned the access of bacteria in colonies to their nutrients, through the assessment of diffusion of solutes inside colonies. The diffusion of high molecular weight molecules was assessed inside Lactococcus lactis colonies grown in a model cheese. The results led to the conclusion that diffusion of molecules inside bacterial colonies depends on the physicochemical properties of the molecules (Floury et al.). In conclusion, since bacteria mainly grow in colonies in foods, increase knowledge on growth and physiology of bacteria growing as colonies is now a crucial issue. This is achieved by the recent development of non-invasive techniques that allows investigating at the microscopic scale with time lapse and quantitative analyses. Knowledge about the growth and the adaptive response of bacteria to the food environment will continue to grow by addressing the remaining questions about interactions between bacterial colonies and their food environment.

Dairy propionibacteria are used as ripening cultures for the production of Swiss-type cheeses, and some strains have potential for use as probiotics. This study investigated the biodiversity of wild dairy Propionibacteria isolates in dairy farms that produce Swiss-type cheeses in Minas Gerais State, Brazil. RAPD and PFGE were used for molecular typing of strains and MLST was applied for phylogenetic analysis of strains of Propionibacterium freudenreichii. The results showed considerable genetic diversity of the wild dairy propionibacteria, since three of the main species were observed to be randomly distributed among the samples collected from different farms in different biotopes (raw milk, sillage, soil and pasture). Isolates from different farms showed distinct genetic profiles, suggesting that each location represented a specific niche. Furthermore, the STs identified for the strains of P. freudenreichii by MLST were not related to any specific origin. The environment of dairy farms and milk production proved to be a reservoir for Propionibacterium strains, which are important for future use as possible starter cultures or probiotics, as well as in the study of prevention of cheese defects.

New approaches, mainly based on mass spectrometry techniques, are being developed and appear as a must in the modern food science and microbiology research to investigate food quality and safety. To date, the investigation of cheese ripening mechanisms has mostly used targeted approaches. The aims of the present project were to assess the use of untargeted metabolomics as an approach to investigate the influence of altering one ripening parameter to generate fine differences in the microbial metabolism within cheese. Two cheeses were made which varied with respect to the spatial distribution of bacterial colonies, leading to cheeses with only big or only small colonies. Liquid chromatography high resolution mass spectrometry metabolic fingerprints were acquired on cheese extracts collected after 2, 13 and 27 days of ripening using two different extraction methods (water or acetonitrile) and analyzed using two different simultaneous ionization modes (positive and negative electrospray). Data processing involving XCMS and multivariate statistical analysis highlighted significant discriminant profiles of the cheese metabolomes according to the two different spatial distributions compared. The different fractions investigated (water and acetonitrile extractions in two ionization modes) were complementary and resulted in a view as global as possible of the cheese metabolome which had been modulated by the spatial distribution of bacterial colonies. Some of the metabolites were then identified using an in-house database. These results show the relevance of cheese LC–HRMS fingerprinting to understand the influence of a ripening parameter generating fine differences on microbial metabolism within cheese.

Lactic acid bacteria (LAB) and propionic acid bacteria (PAB) are widely used in the manufacture of cheeses and other fermented dairy products. Bacterial species used as starters are mainly chosen according to their intrinsic properties: the milk acidifying capacity for LAB starters and the aromatizing properties of PAB, for example. Beyond the general characteristics of a bacterial species, many key phenotypic traits determining their interest for dairy applications depend on the strain within a given species. Through some examples, this review illustrates how the choice of a bacterial strain with specific technological characteristics, within a given species of LAB or PAB, can determine the final properties in the end product. This concerns the technological properties of cheeses, such as flavour, texture, and opening formation, and their functional properties, such as inhibition of undesirable microorganisms and health properties. When known, the genetic determinants of the diversity are presented. This review emphasizes the importance of preserving and exploring microbial resources at the intraspecific level, as an unending source of diversity for innovation in food fermentation.

Fernanda Mozzi, Ph.D., Raúl R. Raya, Ph.D. and Graciela M. Vignolo , Ph.D serve as Editors for the book. They are colleagues at Centro de Referencia para Lactobacilos (CERELA) -CONICET, Tucumán, Argentina. Drs. Mozzi, Raya and Vignolo are engaged as Scientific Researchers by the National (Argentinean) Council for Scientific and Technological Research (CONICET).; Lactic acid bacteria (LAB) have historically been used as starter cultures for the production of fermented foods, especially dairy products. Over recent years, new areas have had a strong impact on LAB studies: the application of omics tools; the study of complex microbial ecosystems, the discovery of new LAB species, and the use of LAB as powerhouses in the food and medical industries. This second edition of Biotechnology of Lactic Acid Bacteria: Novel Applications addresses the major advances in the fields over the last five years. Thoroughly revised and updated, the book includes new chapters. Among them: – The current status of LAB systematics; – The role of LAB in the human intestinal microbiome and the intestinal tract of animals and its impact on the health and disease state of the host; – The involvement of LAB in fruit and vegetable fermentations; – The production of nutraceuticals and aroma compounds by LAB; and – The formation of biofilms by LAB. This book is an essential reference for established researchers and scientists, clinical and advanced students, university professors and instructors, nutritionists and food technologists working on food microbiology, physiology and biotechnology of lactic acid bacteria.

During cheese processing and ripening, bacteria develop as colonies. Substrates and metabolites must then diffuse either from or into the colonies. Exploring how the inner Q1 Q2 cells of the colony access the substrates or get rid of the products leads to study the Q7 Q8 diffusion of solutes inside bacterial colonies immobilized in cheese. Diffusion limitations of substrates within the bacterial colony could lead to starvation for the cells in the center of the colony. This study aimed at better understands ripening at the colony level, by investigating how diffusion phenomena inside colonies vary depending on both the physicochemical properties of the solutes and Lactococcus lactis strain. Dextrans (4, 70, and 155 kDa) and milk proteins (BSA, lactoferrin and aS1-casein) of different sizes and physicochemical properties were chosen as model of diffusing solutes, and two L. lactis strains presenting different surface properties were immobilized as colonies in a model cheese. Diffusion of solutes inside and around colonies was experimentally followed by time-lapse confocal microscopy. Dextran solutes diffused inside both lactococci colonies with a non-significantly different effective diffusion coefficient, which depended mainly on size of the solute. However, whereas flexible and neutral hydrophilic polymers such as dextran can diffuse inside colonies whatever its size, none of the three proteins investigated in this study could penetrate inside lactococci colonies. Therefore, the diffusion behavior of macromolecules through bacterial colonies immobilized in a model cheese did not only depends on the size of the diffusing solutes, but also and mainly on their physicochemical properties. Milk caseins are probably first hydrolyzed by the cell wall proteases of L. lactis and/or other proteases present in the cheese, and then the generated peptides diffuse inside colonies to be furthermetabolized into smaller peptides and amino acids by all the cells located inside the colonies.

The catabolism of amino acids by cheese micro-organisms results in the production of various volatile flavour compounds. It was recently shown to be a rate-limiting factor in the formation of cheese flavour, leading to an increased interest in elucidating the pathways and the flora involved. This paper reviews the ability of propionibacteria (PAB) to produce flavour compounds deriving from branched-chain, aromatic and sulphur-containing amino acids. In culture media, PAB produced volatile compounds derived from Leu, Ile, Met and Phe. In cheese, the presence of PAB is positively correlated to the amount of acids, alcohols and/or aldehydes derived from Leu or Ile. The metabolic pathways of amino acid conversion to flavour compounds by PAB have been only partly elucidated. Aminotransferase(s) catalyse the first step of conversion of branched-chain, aromatic amino acids and methionine, with a higher activity for branched-chain amino acids. The α-keto acids resulting from transamination are further degraded to various compounds by resting cells of PAB. So α-ketoisocaproic acid, derived from Leu, is essentially converted to isovaleric acid by a ketoacid dehydrogenase complex; phenylpyruvic acid, derived from Phe, is converted to phenyllactic acid, phenylacetic acid, benzoic acid and benzaldehyde. Methionine can also be directly degraded by α, γ -elimination, leading to methanethiol. The amino acid catabolism pathways in PAB share similar- ities with those of lactic acid bacteria but PAB seem to produce higher amounts of branched-chain ac- ids, which are important flavour compounds in cheese. propionibacteria / flavour compound / amino acid / catabolism / cheese ripening