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Food transition requires incorporating more plant-based ingredients in our diet, thus leading to the development of new plant-based products, such as yogurt alternatives (YAs). This study aimed at evaluating the impact of lactic acid bacteria (LAB) cocultures and formulation on the physico-chemical and sensory properties of YAs. YAs were made by emulsifying anhydrous milk fat (AMF) or coconut oil in milk and lupin protein suspensions. The starters used, in mono- and cocultures, were the strains

Lactococcus lactis strains are widely used in the dairy industry in fermentation processes for production of cheese and fermented milks. However, the esterolytic activity of L. lactis is not generally considered high. For this reason, purified microbial lipases and esterases are often added in certain dairy processes to generate specific flavors in the final food product. This work demonstrates the superior esterolytic activity of a collection of L. lactis strains isolated from different environmental sources compared with that of dairy-derived strains. It provides further evidence of the more diverse metabolic capabilities displayed by L. lactis strains from environmental sources compared to their domesticated dairy counterparts. Furthermore, the presence of a 1,287-bp gene encoding a 428-amino acid SGNH hydrolase in the high-esterolytic environmental strains suggests a possible link between superior esterolytic activity and the presence of the esterase from the SGNH hydrolase family.

In Algeria

Reducing salt intake can mitigate the prevalence of metabolic disorders. In fermented foods such as cheeses, however, salt can impact the activity of desirable and undesirable microorganisms and thus affect their properties. This study aimed to investigate the effect of salt level on Swiss-type cheese ripening. Since proteolysis is a major event in cheese ripening, three strains of Lactobacillus helveticus were selected on the cell-envelope proteinase (CEP) they harbor. Their proteolytic activity on caseins was studied at six salt levels (0–4.5%) at pH 7.5 and 5.2. Swiss-type cheeses were manufactured at regular, increased, and decreased salt concentrations, and characterized for their composition and techno-functional properties. L. helveticus strains possessed and expressed the expected CEPs, as shown by PCR and shaving experiments. The two strains of L. helveticus that possessed at least the CEP PrtH3 showed the greatest proteolytic activity. Casein hydrolysis in vitro was similar or higher at pH 5.2, i.e., cheese pH, compared to pH 7.5, and slightly decreased at the highest salt concentrations (3.0 and 4.4%). Similarly, in ripened cheeses, these L. helveticus strains showed 1.5–2.4 more proteolysis, compared to the cheeses manufactured without L. helveticus. Regarding the salt effect, the 30% salt-reduced cheeses showed the same proteolysis as regular cheeses, while the upper-salted cheeses showed a slight decrease (−14%) of the non-protein fraction. The microbial and biochemical composition remained unchanged in the 30%-reduced cheeses. In contrast, Propionibacterium freudenreichii, used as ripening bacteria in Swiss cheese, grew more slowly in upper-salted (1.14%, w/w) cheeses, which induced concomitant changes in the metabolites they consumed (−40% lactic acid) or produced (fivefold decrease in propionic acid). Some cheese techno-functional properties were slightly decreased by salt reduction, as extrusion (−17%) and oiling off (−4%) compared to regular cheeses. Overall, this study showed that a 30% salt reduction has little impact in the properties of Swiss-type cheeses, and that starters and ripening cultures strains could be chosen to compensate changes induced by salt modifications in Swiss-type and other hard cheeses.

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

Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the world-wide context of diet diversification.

International audience; Microorganisms grow in concert, both in natural communities and in artificial or synthetic co-cultures. Positive interactions between associated microbes are paramount to achieve improved substrate conversion and process performance in biotransformation and fermented food production. The mechanisms underlying such positive interactions have been the focus of numerous studies in recent decades and are now starting to be well characterized. Lactic acid bacteria (LAB) contribute to the final organoleptic, nutritional, and health properties of fermented food products. However, interactions in LAB co-cultures have been little studied, apart from the well-characterized LAB co-culture used for yogurt manufacture. LAB are, however, multifunctional microorganisms that display considerable potential to create positive interactions between them. This review describes why LAB co-cultures are of such interest, particularly in foods, and how their extensive nutritional requirements can be used to favor positive interactions. In that respect, our review highlights the benefits of co-cultures in different areas of application, details the mechanisms underlying positive interactions and aims to show how mechanisms based on nutritional interactions can be exploited to create efficient LAB co-cultures.

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.

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.

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

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.

– 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.

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.

Lactobacillus helveticus and Propionibacterium freudenreichii are essential starters in Swiss cheesemaking and the release of their intracellular enzymes through autolysis could significantly influence ripening. To provide evidence of this lysis, cheese made from microfiltered thermized milk inoculated with Lb. helveticus ITGLH77, Prop. freudenreichii ITGP23 and a commercial Streptococcus thermophilus was assayed. Starter viability was determined and lysis was monitored during ripening by protein analysis with SDS-PAGE of aqueous cheese extracts and by immunoblot detection of intracellular proteins: dipeptidase (PepD) for Lb. helveticus and methylmalonyl coenzyme A mutase for Prop. freudenreichii. We verified that the species specificity of these lysis markers was towards the cytoplasms of all the species currently used in Swiss cheese. Lb. helveticus exhibited an almost complete loss of viability (99·9%) from the beginning of ripening in the cold room; concomitantly PepD appeared in the cheese extracts and was detected until the end of ripening. Damaged Lb. helveticus cells were also visualized by scanning electron microscopy. In addition, free PepD was also successfully detected in commercial Swiss-related cheeses. All these results clearly demonstrated the autolysis of Lb. helveticus in Swiss cheese. Prop. freudenreichii ITGP23 grew during warm room ripening and no loss of viability was detected after maximal growth (109 cfu/g cheese). Free methylmalonyl-coenzyme A mutase was detected at the end of ripening during cold storage, when the cheese extracts were concentrated 20-fold, demonstrating that the autolysis of Prop. freudenreichii was tardy and limited.

Summary - Prapionibacteria were enumerated using a new medium in 202 raw milk samples taken from tanker lorries, collected over a 1-year period fram 9 regions of France. This medium, called Pal Propiobac, has been compared with yeast extract lactate agar (YELA) for its efficiency to enumerate propionibacteria in 29 of these milk samples. Prapionibacteria counts were of the same order of magnitude on both media. However, total counts on YELA (reflecting ail the milk flora able to graw on the medium) were markedly higher than total counts on Pal Propiobac, which shows a higher selectivity of the latter medium towards milk microorganisms other than propionibacteria. Moreover, more than onequarter (8 of 29) of the milk samples could not have been enumerated on YELA because of the lack of colonies of typical appearance on this medium. Prapionibacteria counts in raw milk ranged between less than 10 cfu/ml to 1.3 104 cfu/ml. The variations observed for milk samples collected the same day in a given region were of the same order of magnitude as variations depending on regional or seasonal factors. The geometric mean of propionibacteria counts of ail the tested samples was 2.19 log cfu/ml, with a standard deviation of 0.58. The significance of the propionibacteria content of milk for cheese technology is discussed.

Dairy propionibacteria are anaerobic to aerotolerant mesophilic bacteria found in dairy products. They are Gram-positive, high G + C, pleomorphic rods. They include the species Propionibacterium freudenreichii, P. acidipropionici, P. jensenii, P. thoenii, P. cyclohexanicum, and P. microaerophilum. They can be isolated from various ecological niches, including soil, rumen, and dairy plants, consistently with their remarkable adaptive mechanisms. Their peculiar obligatory fermentative metabolism leads to the formation of propionic acid as a major end product from a large range of substrates. Propionibacterium freudenreichii is used as ripening culture in Emmental and related cheeses. It grows during the ripening and ferments lactic acid produced by lactic acid bacteria to propionic acid, acetic acid, H2O, and CO2, leading to the characteristic flavor and eyes of Emmental and related varieties. It also releases free fatty acids, from milk fat lipolysis, and short branched-chain fatty acids involved in cheese flavor, and other valuable products including vitamins and conjugated linoleic acid. Propionibacteria, when used as probiotics, were shown to beneficially modulate intestinal microbiota, enzymatic activities, and motility. Genomic tools, including inactivation and expression vectors, have been developed and the genome of P. freudenreichii (2.6 Mb) has been sequenced. The postgenomic era will indeed improve understanding and mastering their applications, both in food products transformation/preservation and in health applications.

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.

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 103 copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese.

Indigenous yeasts can be detected at high populations in raw milk Cantal cheese, a French Protected Denomination of Origin (PDO) hard cheese. To investigate their use as adjunct cultures to promote flavour development in Cantalet (small Cantal) cheese, three strains isolated from raw milk Cantal cheese, Kluyveromyces lactis, Yarrowia lipolytica, and Pichia fermentans were added at 3 (E3) and 5 (E5) log(10) colony-forming units (cfu)/mL to microfiltered milk at a ratio of 80/10/10 viable cells, respectively. The global microbial, compositional and biochemical changes induced by the presence of yeasts in cheese were determined. Adjunct yeasts did not grow but stayed at viable populations of approximately 4 and 6 log(10) cfu/g in E3 and E5 cheeses, respectively, throughout the ripening period. They were mainly constituted of K. lactis, while P. fermentans and Y. lipolytica were not detectable after 3 and 45 days of ripening, respectively. Several species of indigenous yeasts were also detected in E3 cheeses at the beginning of ripening only, and in the control cheeses without yeasts added. Lactoccoci survived for longer periods in the presence of yeast adjuncts, while, conversely, the viability of Streptococcus thermophilus decreased more rapidly. The addition of yeasts did not influence cheese composition and total free amino acid content. In contrast, it slightly increased lipolysis in both E3 and E5 cheeses and markedly enhanced the formation of some volatile aroma compounds. The concentrations of ethanol, ethyl esters and some branched-chain alcohols were 6 to 10 fold higher in E5 cheeses than in the control cheeses, and only slightly higher in E3 cheeses. This study shows that K. lactis has a potential as cheese adjunct culture in Cantalet cheese and that, added at populations of 4-5 log(10) cfu/g cheese, it enhances the formation of flavour compounds.

International audience; Proteolysis is a major event during cheese ripening. In Emmental cheese, the contribution of plasmin and thermophilic lactic starters to proteolysis has been well established. Our aim was to assess the contribution of each ripening flora to secondary proteolysis. With this aim, we used the aqueous phase (or juice) of Emmental cheese as a culture medium, which contains the enzymes released by thermophilic starters. Crude juice (containing all cheese flora) and cell-free juice either inoculated with two propionibacteria (PAB) strains or uninoculated (control), were incubated under anaerobiosis at 24 °C for 20 d. Free amino groups and free amino acids, bacterial growth and organic acids were followed throughout the incubation time. Facultatively heterofermentative lactobacilli (FHL) represented the main flora growing in crude juice up to 10 d incubation. FHL and PAB respectively reached 4.0 $\times$ 10$^8$ and 1.0 $\times$ 10$^{10}$ cfu$\cdot$mL$^{-1}$ of juice and consumed citrate and lactate, in accordance with that already observed in cheese. The level of amino groups increased linearly during incubation time by a factor of 1.9 in crude juice, 1.7 in cell-free juice and only 1.5 in juice inoculated with PAB strains, showing that the enzymes of thermophilic starters are the main contributors to secondary proteolysis. FHL growth resulted in a 16% increase in free amino acids compared to cell-free juice and in exhaustion of Arg and His. Both PAB strains caused a 18-27% decrease in free amino acids and also modified the amino acid distribution, with consumption of Asp, Gly, Glu, Ser and Ile.; Les lactobacilles hétérofermentaires facultatifs et les bactéries propioniques ont une faible contribution à la protéolyse secondaire de l'Emmental. La protéolyse est un phénomène majeur dans l'affinage du fromage. Dans l'Emmental, la contribution de la plasmine et des levains thermophiles à la protéolyse secondaire a été bien établie. Le but de cette étude était de déterminer la contribution de chacune des flores d'affinage à la protéolyse secondaire. Pour cela, nous avons utilisé de la phase aqueuse d'Emmental (ou jus), qui contient les enzymes relarguées par les levains thermophiles lors de leur lyse, comme milieu de culture. Le jus de fromage brut (contenant toutes les flores du fromage), le jus exempt de cellules bactériennes utilisé soit tel quel (témoin), soit ensemencé avec deux souches de bactéries propioniques (PAB) ont été incubés en anaérobiose à 24 °C pendant 20 j. Les groupements aminés libres et les acides aminés libres ainsi que les paramètres de croissance et de fermentation ont été suivis durant l'incubation. Les lactobacilles hétérofermentaires facultatifs (FHL) représentaient la flore principale poussant dans le jus brut jusqu'à dix jours d'incubation. Les FHL et les PAB atteignaient respectivement 4,0 $\times$ 10$^8$ and 1,0 $\times$ 10$^{10}$ ufc$\cdot$mL$^{-1}$ de jus et consommaient le lactate et le citrate, en accord avec les résultats déjà observés dans le fromage. Les taux de groupements amines libres augmentaient linéairement pendant l'incubation par un facteur 1,9 dans le jus brut, 1,7 dans le jus témoin et seulement 1,5 dans le jus ensemencé avec les souches de PAB, montrant que les enzymes des levains thermophiles contribuent majoritairement à la protéolyse secondaire. La croissance des FHL résultait en un accroissement de 16 % des acides aminés libres par rapport au jus témoin, et une disparition de Arg et His. Chacune des deux souches de PAB entraînait une chute de 18-27 % en acides aminés libres et modifiait également la distribution en acides aminés, en consommant Asp, Gly, Glu, Ser et Ile.

International audience; Lactic acid bacteria (LAB) are largely used in coculture in food fermentations. The use of different LAB species or strains as starter can bring complementary functionalities participating to the overall quality of fermented food. Fermentation can benefit from LAB’s interactions, resulting in a more efficient fermentation. Few is known about positive LAB’s interactions that are beneficial to fermentation processes. Lactobacillus delbrueckii CIRM-BIA865 (Ld865) and Lactiplantibacillus plantarum CIRM-BIA777 (Lp777) are two strains able to ferment soy juice (SJ). In SJ monocultures, Ld865 was obligately homolactic and released 17 amino acids while Lp777 was facultatively heterofermentative and consumed 10 of the 17 amino acids released by Lp777. When these strains were cocultivated in SJ, the fermentation time was reduced of 30% compared to monocultures. Furthermore, Ld865 cells were four times shorter in coculture than in monoculture and Ld865 population increased tenfold. In this work, we aimed to reveal how Lp777 and Ld865 interact with each other to improve SJ fermentation. Thanks to transcriptomic and metabolic analyses carried out along the kinetic of fermentation, we compared the adaptations of their metabolisms in coculture versus in monoculture. Transcriptomics data showed, among others, that Lp777 induced the expression of genes involved in the regulation of oxidation-reduction potential at a higher level in coculture than in monoculture. The oxidation-reduction potential measured in cocultured SJ was intermediate between those measured in monocultures. The better regulation of the oxidation-reduction potential in coculture seems to involve methionine released by Ld865. Lp777 and Ld865 could also cooperate to metabolize carbon. In conclusion, LABs with distinct and complementary metabolic profiles can be useful to take over SJ fermentation. This work opens new avenues for strains selection intended to cooperate in plant-based fermentation. Several examples of positive LAB’s interactions that are beneficial in SJ fermentation will be provided during this communication.

International audience; In cheese production, lactic acid bacteria (LAB) and propionic bacteria are key players in the production of metabolites that confer nutritional and organoleptic qualities. However, the contribution of each species to the final quality of cheeses is not fully elucidated. The objective was to determine which species contribute to acidify and produce flavour compounds, by which metabolic pathways, according to which temporality and also to investigate the bacterial metabolic interactions contributing to the functioning of the cheese ecosystem.We sequenced and annotated: Lactococcus lactis subsp. lactis biovar diacetylactis CIRM-BIA1206 (LL), Lactiplantibacillus plantarum CIRM-BIA465 (LP), Propionibacterium freudenreichii CIRM-BIA122 (PF). We reconstructed the metabolic pathways and developed a community metabolic model. Four semi-hard cheeses were made with LL, LP and PF and were analyzed throughout manufacturing. Bacteria, sugars, organic acids and flavour compounds were quantified and RNA sequenced.The analysis of differentially expressed genes showed at which moment of the manufacturing process the genes involved in the catabolism of lactose and citrate and in the synthesis pathways of different flavour compounds: lactic, acetic, propionic, isovaleric (old cheese flavour), acetoin (cheesy flavour) acids were induced. Lactose catabolism was induced in LP and PF (Leloir pathway) during acidification and then in LL (tagatose pathway) during ripening. Acetoin production was induced in LL, LP and PF from the beginning of the manufacturing process to the beginning of ripening. The synthesis of propionic and isovaleric acids were attributed to PF and the corresponding metabolisms were induced from the beginning of the manufacturing then stably expressed during the first month of the ripening. Since the mixed fermentation pathways were induced during ripening, acetic acid was likely produced by LL, LP and PF at this stage. Implementation of the metabolic community model in the Smetana tool revealed the molecular basis of the previously discussed commensalism between LAB and PF. LAB produce lactic acid and potentially ribose, succinate, glycerol, serine and phenylalanine for the benefit of PF. These interactions, identified in silico, remain to be validated in vitro.All these results make metatranscriptomics associated with metabolic models, tools of choice to better understand and control the metabolisms and interactions governing the functioning of cheese ecosystems.

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

5 tables 7 graph.; National audience

Article à destination des communautés scientifiques publiés dans une revue à comité de lecture; Dear colleagues, members of the Editorial Board, and reviewers of Dairy Science & Technology,This is the last issue of Dairy Science & Technology.Overall, more than 3000 papers have been published since 1921, under the new or the former title Dairy Science & Technology and Le Lait, as well as Special Issues containing papers presented at key symposia of the International Dairy Federation and other international symposia. The whole collection of papers published since 1921 has been digitized in totality and is available in the open archive Hal at https://hal.archivesouvertes.fr/ARINRA-DST and https://hal.archives-ouvertes.fr/ARINRA-LELAIT. This constitutes a testimony of an inestimable value over almost a century of research in dairy science and technology, where one can measure, for example, how research questions are cyclic and the way to write has totally changed. See, for example, thisarticle written by Dr. Violle of the Institut Pasteur in 1921 entitled Les Microbes et le Lait(Microbes and Milk) at https://hal.archives-ouvertes.fr/hal-00894664/ or this paper from M. Moore in 1922 entitled Le troupeau de vaches le plus productif du monde et comment a-t il été constitué (The most productive herd of cows in the world and how it was formed) at https://hal.archives-ouvertes.fr/hal-00894688/. I have worked as the editor-in-chief since 2008, with the valuable and essential support of Magalie Weber, as managing editor. I have learned a lot through this mandate about the world of journal editing, but it is time now for me to leave this job to take on new missions. I would like to warmly thank the numerous scientists who gave their time to the journal as board members or as reviewers, to assist us with the hard tasks of prescreening and reviewing manuscripts.Best regards,Anne ThierryEditor-in-chief