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

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the "Tanche" olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had β-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

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

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.

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.

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

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.

Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, including modulation of bifidobacteria, colon epithelial cell proliferation and apoptosis, and intestinal inflammation, rely on active metabolism in situ . Survival and activity are thus key factors determining its efficacy, creating stress adaptation and tolerance bottlenecks for probiotic applications. Growth media and growth conditions determine tolerance acquisition. We investigated the possibility of using sweet whey, a dairy by-product, to sustain P. freudenreichii growth. It was used at different concentrations (dry matter) as a culture medium. Using hyperconcentrated sweet whey led to enhanced multistress tolerance acquisition, overexpression of key stress proteins, and accumulation of intracellular storage molecules and compatible solutes, as well as enhanced survival upon spray drying. A simplified process from growth to spray drying of propionibacteria was developed using sweet whey as a 2-in-1 medium to both culture P. freudenreichii and protect it from heat and osmotic injury without harvesting and washing steps. As spray drying is far cheaper and more energy efficient than freeze-drying, this work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness. IMPORTANCE In this study, we demonstrate that sweet whey, a dairy industry by-product, not only allows the growth of probiotic dairy propionibacteria, but also triggers a multitolerance response through osmoadaptation and general stress response. We also show that propionibacteria accumulate compatible solutes under these culture conditions, which might account for the limited loss of viability after spray drying. This work opens new perspectives for more energy-efficient production of dairy starters and probiotics.

Dairy propionibacteria display probiotic properties which require high populations of live and metabolically active propionibacteria in the colon. In this context, the probiotic vector determines probiotic efficiency. Fermented dairy products protect propionibacteria against digestive stresses and generally contain a complex mixture of lactic and propionic acid bacteria. This does not allow the identification of dairy propionibacteria specific beneficial effects. The aim of this study was to develop a dairy product exclusively fermented by dairy propionibacteria. As they grow poorly in milk, we determined their nutritional requirements concerning carbon and nitrogen by supplementing milk ultrafiltrate (UF) with different concentrations of lactate and casein hydrolysate. Milk or UF supplemented with 50 mM lactate and 5 g L−1 casein hydrolysate allowed growth of all dairy propionibacteria studied. In these new fermented dairy products, dairy propionibacteria remained viable and stress-tolerant in vitro during minimum 15 days at 4 °C. The efficiency of milk fermented by the most tolerant Propionibacterium freudenreichii strain was evaluated in piglets. Viability and SCFA content in the colon evidenced survival and metabolic activity of P. freudenreichii. This work results in the design of a new food grade vector, which will allow preclinical and clinical trials.

In complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS.

The actual impact of the somatic cells in the dairy technology is still ill-defined, because the increase in milk somatic cell count, usually correlated with mastitis factors, impairs the raw milk composition, through mainly unwanted proteolysis and lipolysis. This study used microfiltration technologies for recovering high quantity of somatic cells and for clarifying their role in cheese quality. Three series of Swiss-type cheeses were manufactured by adding 0 (control), 4 × 105 and 9 × 105 somatic cells mL−1. These cells were traced for the first time during the cheese making process by using adapted flow cytometry and real-time quantitative PCR. Proteolysis and lipolysis indices were measured throughout ripening time. Only a weak increase in lipolysis (+28%) and proteolysis (+8%) was observed in the highest somatic cell count cheese, despite 73% of the cells trapped within the cheeses. Our approach gives a new view of somatic cell role in cheese milk alteration.

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, 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.

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

Propionibacterium freudenreichii is widely used in Swiss-type cheese manufacture, where it contributes to flavour and eye development. It is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit− and lac−/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac−/nit−) has also been reported. The aim of this study was to revisit the relevance of the subdivision of P. freudenreichii into subspecies, by confirming the existence of unclassifiable strains. Relevant conditions to test the ability of P. freudenreichii for lactose fermentation and nitrate reduction were first determined, by using 10 sequenced strains, in which the presence or absence of the lactose and nitrate genomic islands were known. We also determined whether the subdivision based on lac/nit phenotype was related to other phenotypic properties of interest in cheese manufacture, in this case, the production of aroma compounds, analysed by gas chromatography-mass spectrometry, for a total of 28 strains. The results showed that a too short incubation time can lead to false negative for lactose fermentation and nitrate reduction. They confirmed the existence of four lac/nit phenotypes instead of the two expected, thus leading to 13 unclassifiable strains out of the 28 characterized (7 lac+/nit+ and 6 lac−/nit−). The production of the 15 aroma compounds detected in all cultures varied more within a lac/nit phenotype (up to 20 times) than between them. Taken together, these results demonstrate that the division of P. freudenreichii into two subspecies does not appear to be relevant.

Aim was to elucidate the effect of interspecies and intraspecies diversity of dairy propionibacteria on the production of aroma compounds in a cheese context. Flavor is an important sensory property of fermented food products, including cheese, and largely results from the production of aroma compounds by microorganisms. Propionibacterium freudenreichii is the most widely used species of dairy propionibacteria ; it has been implicated in the production of a wide variety of aroma compounds through multiple metabolic pathways and is associated with the flavor of Swiss cheese. However, the ability of other dairy propionibacteria to produce aroma compounds has not been characterized. This study sought to elucidate the effect of interspecies and intraspecies diversity of dairy propionibacteria on the production of aroma compounds in a cheese context. A total of 76 strains of Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, and Propionibacterium acidipropionici were grown for 15 days in pure culture in a rich medium derived from cheese curd. In addition, one strain each of two phylogenetically related non-dairy propionibacteria, Propionibacterium cyclohexanicum and Propionibacterium microaerophilum were included. Aroma compounds were analyzed using headspace trap-gas chromatography-mass spectrometry (GC–MS). An analysis of variance performed on GC–MS data showed that the abundance of 36 out of the 45 aroma compounds detected showed significant differences between the cultures. A principal component analysis (PCA) was performed for these 36 compounds. The first two axes of the PCA, accounting for 60% of the variability between cultures, separated P. freudenreichii strains from P. acidipropionici strains and also differentiated P. freudenreichii strains from each other. P. freudenreichii strains were associated with greater concentrations of a variety of compounds, including free fatty acids from lipolysis, ethyl esters derived from these acids, and branched-chain acids and alcohols from amino acid catabolism. P. acidipropionici strains produced less of these compounds but more sulfur-containing compounds from methionine catabolism. Meanwhile, branched-chain aldehydes and benzaldehyde were positively associated with certain strains of P. jensenii and P. thoenii. Moreover, the production of compounds with a common origin was correlated. Compound abundance varied significantly by strain, with fold changes between strains of the same species as high as in the order of 500 for a single compound. This suggests that the diversity of dairy propionibacteria can be exploited to modulate the flavor of mild cheeses.

The headspace compounds of Emmental aqueous phase (juice) and oil were analysed during ripening by dynamic headspace-gas-chromatography–mass spectrometry. We first checked that peak areas varied linearly with concentrations by using a standard addition method for 9 compounds. The slopes of linear regression curves were 1–1800 times higher for juice than for oil, indicating that these compounds were more easily released from juice. Consequently, although most flavour compounds are more concentrated in cheese oil than in cheese juice, they were similarly recovered from the headspace of both types of samples. Eighty-two compounds were identified, 70% of them being found in both juice and oil. Alcohols and esters markedly increased in number and concentration, essentially during the ripening in the warm room, as well as, to a lesser extend, sulphur compounds, methyl ketones, and 3-methylbutanal, whereas the other aldehydes decreased.

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis. Fil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Falentin, Hélène . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Maillard, Marie-Bernadette . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Chuat, Victoria . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Medina, Roxana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina Fil: Parayre, Sandrine . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Thierry, Anne. INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia

In complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS.

Metabolic fingerprinting is an untargeted approach which has not yet been undertaken to investigate cheese. This study is a proof of concept, concerning the ability of mass spectrometry (MS) metabolic fingerprinting to investigate modifications induced by bacterial metabolism in cheese over time. An ultrafiltrated milk concentrate was used to manufacture model cheeses inoculated with Lactococcus lactis LD61. Metabolic fingerprints were acquired after 0, 8 and 48 h from two different fractions of the metabolome: the water-soluble fraction using liquid chromatography–high resolution-MS and a volatile fraction using gas chromatography–MS. Metabolic fingerprints differed significantly over time. Forty-five metabolites were identified, including well-known cheese metabolites, such as 12 amino acids and 25 volatile metabolites, and less studied ones, such as four vitamins, uric acid, creatine and L-carnitine. These results showed the relevance of cheese MS fingerprinting to generate new findings and to detect even slight differences between two conditions.

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Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It grows when cheeses are ripened in a warm room (about 24°C). Cheeses with an acceptable eye formation level are transferred to a cold room (about 4°C), inducing a marked slowdown of propionic fermentation, but P. freudenreichii remains active in the cold. To investigate the P. freudenreichii strategies of adaptation and survival in the cold, we performed the first global gene expression profile for this species. The time-course transcriptomic response of P. freudenreichii CIRM-BIA1(T) strain was analyzed at five times of incubation, during growth at 30°C then for 9 days at 4°C, under conditions preventing nutrient starvation. Gene expression was also confirmed by RT-qPCR for 28 genes. In addition, proteomic experiments were carried out and the main metabolites were quantified. Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from 30°C to 4°C (P1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1(T) strain over-expressed genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis, and in glycogen synthesis. Interestingly, the expression of different genes involved in the formation of important cheese flavor compounds, remained unchanged at 4°C. This could explain the contribution of P. freudenreichii to cheese ripening even in the cold. In conclusion, P. freudenreichii remains metabolically active at 4°C and induces pathways to maintain its long-term survival.

Seven Propionibacterium freudenreichii strains exhibited similar responses when placed at 4°C. They slowed down cell machinery, displayed cold stress responses, and rerouted their carbon metabolism toward trehalose and glycogen synthesis, both accumulated in cells. These results highlight the molecular basis of long-term survival of P. freudenreichii in the cold.

Modifying the lipid composition of milk and dairy products, to improve their nutritional properties, without negatively altering their technological, sensorial and functional qualities, constitutes a challenge for the dairy sector. This study was performed to increase the polyunsaturated fatty acids (PUFA) and decrease the saturated fatty acids (SFA) content of goats’ milk, under real field conditions and production, by means of altering the animals diet. The effect of these changes were characterised during the manufacture of soft ripened cheese and assessing the impact on physicochemical and sensorial characteristics. Two groups of 30 crossbreed Alpine dairy goats were fed either a control diet or a diet supplemented with oilseeds providing 50.4 g UFA.goat−1.day−1 or 69.6 g UFA.goat−1.day−1 which supplied respectively 1.4 and 2.4 % of alpha-linolenic acid (ALA). Supplementing the feed of dairy goats with PUFA was shown to have beneficial effects on the FA composition of goat’s milks and cheeses: (1) an increase in the content of ALA (18:3n-3; from 0.78 to 1.78 g.100 g−1 of total FA) and rumenic acid (main CLA; from 0.75 to 1.27 g.100 g−1 of total FA); (2) a decrease in the n-6/n-3 PUFA ratio (from 4.6 to 2) and the overall SFA content (from 66 to 60.4 g.100 g−1 of total FA). The corrected cheese yield was higher for the supplemented milks (16.3 %) in comparison to the control milks (15.4 %). Both cheeses showed a similar evolution in the levels of proteolysis and lipolysis with no flavour defects being detected. Cheese sensory scores for the two types of cheeses were similar. Hence, healthier goats’ milk and cheese FA profiles were obtained with good sensorial characteristics.

High-throughput sequencing technologies have the potential to decipher genomic diversity at a moderate cost. Propionibacterium freudenreichii is an Actinobacterium used in cheese technology (responsible for aroma and holes in Swiss cheese) and that has probiotic properties (bifidogenic and with anti-inflammatory properties in human and animal). 23 strains of Propionibacterium freudenreichii covering 18 different sequence types out of 46 (Dalmasso et al. AEM 2010) were (i) sequenced by Solexa-Illumina paired-end sequencing and (ii) de novo assembled using Velvet software. Depending on strain, 63 to 166 scaffolds were obtained. These scaffolds were arranged along the reference genome CIRM-BIA1, previously obtained from traditional Sanger sequencing (Falentin et al. PlosOne 2010). For each strain, single nucleotide polymorphism and insertion deletion events were plotted against reference genome. Sequences were automatically annotated (by blastp and pattern matching) on the INRA AGMIAL platform. By bidirectional best-hit, each gene was attributed either to core genome or accessory genome. In some strains, some genes belonging to the accessory genome corresponded with genomic islands and confered peculiar phenotypes (ability to lactose degradation for exemple). In the future, screening of P. freudenreichii (~500 strains) collection on genetic basis at CIRM-BIA biological resource center (Rennes) will help in strain choice for cheese starter and probiotic market.

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Controlling lipolysis in cheese is necessary to ensure the formation of desirable flavor. To get a better understanding of the mechanism of lipolysis in Swiss cheese, cheeses were manufactured with and without (control) the addition of Propionibacterium freudenreichii. Products of lipolysis were quantified throughout ripening. Half of the free fatty acids (FFA) released in milk (3.66 mg/g fat), in particular the short-chain FFA, were lost in the whey during curd drainage, whereas diglycerides and monoglycerides were retained within the curd. P. freudenreichii was responsible for the release of most FFA during ripening (10.84 and 0.39 mg/g fat in propionibacteria-containing and control cheeses, respectively). Indices of lipolysis displayed low specificity. All types of FFA were released, but butyric and palmitic acids more significantly, which could be due to a low sn-1,3 regioselectivity. All glycerides were hydrolyzed in the following order: monoglycerides > diglycerides > triglycerides. The results of this study show the quantitative and qualitative contributions of the different lipolytic agents to Swiss cheese lipolysis.

Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii , a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii . Since cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico . The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico , biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by P. freudenreichii .

BackgroundPropionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027(T)), was sequenced with an 11-fold coverage.Methodology/principal findingsThe circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters.Conclusions/significanceWith the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.

Aims: Short branched-chain fatty acids (BCFAs) are cheese flavour compounds, which result from the conversion of branched-chain amino acids (BCAAs). In Swiss cheese, the production of short BCFAs is mainly performed by Propionibacterium freudenreichii and is strain dependent. Our aim was to investigate the possible links between the biosynthesis of short BCFAs and membrane BCFAs in P. freudenreichii. Methods and Results: Short and membrane BCFAs were analysed by gas chromatography- mass spectrometry. Two strains differing in their capacities to release short BCFAs were selected. Tri-deuterated-labelled leucine was used in both strains as a precursor of short extracellular iso-BCFAs and of membrane iso-BCFAs. The proportions of anteiso : iso BCFAs synthesized varied as function of the BCAAs provided in the growth medium, from 72 : 28 to 100 : 0, with leucine and valine, and with isoleucine as sole BC precursors, respectively. The branching pattern of short BCFAs exactly matched that of membrane BCFAs, whatever the exogenous BCAAs provided. Conclusions: The biosynthesis of short BCFAs is closely related to that of membrane BCFAs in P. freudenreichii. Significance and Impact of the Study: The biosynthesis of short BCFAs in P. freudenreichii depends more on the strain than on the presence of exogenous BC precursors.

Esters are common flavour compounds of cheese. Our aim was to investigate the impact of esters on the flavour of Swiss cheese. Swiss cheeses were manufactured without (controls) or with (E) addition of ethanol to induce variations in ethyl ester concentrations. Cheese flavour was characterized by quantitative and qualitative (frequency of perception of 53 attributes) sensory evaluation. E cheeses contained ∼9 times more ethanol (137 μg g −1 ) and ∼30 times more ethyl esters (26–172 ng g −1 ) than the controls. All cheeses had a typical Swiss cheese flavour. However, E cheeses were characterized by greater frequencies of perception of ‘apricot/peach’, ‘caramel’, ‘pear’, and ‘pepper’ odours, ‘walnut’ aroma, and a lower frequency of ‘soap’ odour and ‘cooked cabbage’ aroma. These results confirm that ethanol limits ethyl ester synthesis in Swiss cheese, show that moderate concentrations of ethanol are sufficient to induce perceptible flavour changes, and open new possibilities to diversify cheese flavour.

This study followed the progression of lipolysis in Emmental cheese by quantifying the concentrations of individual free fatty acids (FFA) released during ripening in each of the different rooms: 12 days at 12 degrees C, 28 days at 21 degrees C, and 8 days at 4 degrees C. Lipolysis, which corresponded to 1.56% of fat, mainly occurred in the 21 and 4 degrees C rooms, with 68 and 16.5% of total FFA, respectively. The nonselectivity of lipolytic enzymes was evidenced: all fatty acids were released with level of > or =1%. Differential scanning calorimetry experiments showed that the thermal properties of cheese were affected by (i) lipolysis of fat, that is, the monoacylglycerols, diacylglycerols, and FFA that may be localized at the fat/whey interface, and/or by (ii) hydrolysis of high-melting-point triacylglycerols constituted mainly by long-chain saturated fatty acids (e.g., palmitic acid). Analysis of the cheese microstructure was performed using confocal laser scanning microscopy. Fat globules were mainly disrupted after pressing of curd grains, leading to the release of the milk fat globule membrane (MFGM); fat inclusions were surrounded by pockets of whey, delimited by casein strands. Moreover, colonies of bacteria were preferentially localized in situ at the fat/protein interface. This study showed that both the localization of bacteria and the supramolecular organization of fat which was not protected by the MFGM can help the accessibility of milk fat to lipolytic enzymes and then contribute to the quality of cheese.