First Insight into the Technological Features of Lactic Acid Bacteria Isolated from Algerian Fermented Wheat Lemzeiet

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

New insights about phenotypic heterogeneity within Propionibacterium freudenreichii argue against its division into subspecies

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.Fil: de Freitas, Rosangela. Universidade Federal de Viçosa. Departamento de Tecnologia de Alimentos; Brasil. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Madec, Marie Noelle. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Chuat, Victoria. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Maillard, Marie Bernadette. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Falentin, Hélène. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Carvalho, Antonio Fernandes de. Universidade Federal de Viçosa. Departamento de Tecnologia de Alimentos; BrasilFil: Valence, Florence. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; FranciaFil: Thierry, Anne. Institut National de la Recherche Agronomique; Francia. Science et Technologie du Lait et de l; Franci

A long and abundant non-coding RNA in Lactobacillus salivarius

Lactobacillus salivarius, found in the intestinal microbiota of humans and animals, is studied as an example of the sub-dominant intestinal commensals that may impart benefits upon their host. Strains typically harbour at least one megaplasmid that encodes functions contributing to contingency metabolism and environmental adaptation. RNA sequencing (RNA-seq) transcriptomic analysis of L. salivarius strain UCC118 identified the presence of a novel unusually abundant long non-coding RNA (lncRNA) encoded by the megaplasmid, and which represented more than 75 % of the total RNA-seq reads after depletion of rRNA species. The expression level of this 520 nt lncRNA in L. salivarius UCC118 exceeded that of the 16S rRNA, it accumulated during growth, was very stable over time and was also expressed during intestinal transit in a mouse. This lncRNA sequence is specific to the L. salivarius species; however, among 45 L. salivarius genomes analysed, not all (only 34) harboured the sequence for the lncRNA. This lncRNA was produced in 27 tested L. salivarius strains, but at strain-specific expression levels. High-level lncRNA expression correlated with high megaplasmid copy number. Transcriptome analysis of a deletion mutant lacking this lncRNA identified altered expression levels of genes in a number of pathways, but a definitive function of this new lncRNA was not identified. This lncRNA presents distinctive and unique properties, and suggests potential basic and applied scientific developments of this phenomenon

Caractérisation et conservation de la diversité bactérienne d’un lait fermenté traditionnel breton, le Gwell en lien avec la préservation d’une race locale de vache, la Bretonne Pie Noir

Le Gwell est un lait fermenté traditionnel spécifique de la Bretagne. Il est obtenu à partir de lait de vaches de race Bretonne Pie Noir, inoculé avec une portion de la fabrication précédente (appelé ferment) sans aucun recours à des levains commerciaux. Les productions de Gwell partagent une texture ferme et onctueuse et un gout frais et acidulé, avec des caractéristiques organoleptiques propres à chaque producteur. Les producteurs sont malheureusement parfois confrontés à la perte de leur ferment et doivent alors avoir recours à la solidarité d’autres producteurs pour réacquérir un ferment opérationnel. Ces pertes de ferments sont un frein au développement de la production de Gwell et donc à la valorisation de lait issu de vaches Bretonne Pie Noir. Cette race emblématique de la Bretagne, caractérisée par une rusticité hors du commun et un lait très riche en matière grasse totalisait au milieu du 19ème siècle près de 900 000 têtes. La modernisation des pratiques agricoles alliée à une orientation productiviste forte a conduit à une quasi extinction de l’espèce, ce qui a conduit à initier en 1976 un programme de sauvegarde de l’espèce. Le nombre de vaches s’élève ainsi aujourd’hui à près de 2500 femelles. La transformation du lait en Gwell est, pour les éleveurs, un moyen de valoriser la qualité du lait de Bretonne Pie Noir en conservant sa valeur ajoutée. Les éleveurs qui transforment le lait en Gwell œuvrent ainsi à la sauvegarde de l’espèce Bretonne Pie Noir, mais aussi à la préservation de la diversité microbienne, du patrimoine et des savoir-faire paysans associés. La caractérisation de l’écosystème microbien du ferment Gwell, pour mieux maitriser sa conservation et sécuriser ainsi la production de Gwell, participe de ce fait au maintien de la race Bretonne Pie Noir. Dans ce contexte notre étude visait à caractériser l’écosystème microbien du Gwell pour sécuriser les souches à l’origine de la typicité du produit. Nous avons ainsi montré que toutes les productions de Gwell avaient une flore bactérienne dominante similaire, composée de deux sous-espèces de la bactérie lactique Lactococcus lactis (subsp. lactis et subsp. cremoris). En fonction des producteurs, le nombre de souches de chaque sous-espèce peut varier avec dans certain cas la présence de Streptococcus thermophilus. De plus, nous avons identifié et caractérisé des souches spécifiques à chaque producteur et montré une forte résilience de l’écosystème pouvant expliquer en partie les différences organoleptiques observées entre les Gwell de différents producteurs

A Temporal -omic Study of Propionibacterium freudenreichii CIRM-BIA1T Adaptation Strategies in Conditions Mimicking Cheese Ripening in the Cold

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-BIA1T 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 (P<0.05 and |fold change|>1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1T 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

Pulsed-Field Gel Electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides

International audienc

Les automates de phénotypage à haut débit du CIRM

International audienceLe Centre International de Ressources Microbiennes (CIRM) est un Groupe ment d’Intérêt Scientifique (GIS), créé en 2004 par l’INRA, qui a pour vocation la préservation, l’enrichissement et la valorisation de la diversité microbienne présente dans ses collections. Il compte aujourd’hui plus de 22 000 souches de champignons filamenteux, levures et bactéries. Les collections du CIRM ont la particularité d’être spécialisées, les ressourcesbiologiques hébergées étant liées à des thématiques précises : des Levures d’intérêt biotechnologique, des Bactéries d’intérêt alimentaire, pathogènes des animaux et de l’Homme, associées aux Plantes, et des Champignons Filamenteux d’intérêt agro-industriel. Ces collections sont capables d’offrir plus d’une centaine de souches d’une seule et même espèce. Elles représentent ainsi un vivier très intéressant pour la recherche de fonctionnalités d’intérêts dans les domaines de l’agroalimentaire, de la santé et des biotechnologies. Pour investiguer les capacités de ses ressources microbiennes, le GIS CIRM s’est doté de deux outils de criblage, l’un au CIRM dédié aux Bactéries d’Intérêt Alimentaire (CIRM-BIA) de Rennes, et l’autre au CIRM dédié aux ChampIgnons Filamenteux (CIRM-CF) à Marseille, chaque plateforme étant optimisée pour la manipulation des ressources biologiques lui étant propres

Pulsed Field Gel Electrophoresis for Dairy Propionibacteria

International audienc

Proteomic approach for bacteria of food interest identification: on-plate trypsinolysis followed by MALDI-MS/MS

Whatever the field (clinical or environmental), classification and identification of bacterial strains is fundamental. For fermented foods industries, bacterial identification is a key element for risk assessment. Bacteria of food interest, mainly represented by lactic acid bacteria, are a major part of many fermented products and are found in a variety of environments. Thus the development of a rapid and simple method for their identification is particularly relevant. Most of the techniques currently used are based on molecular approaches or conventional carbohydrates use tests. However, these methods are generally time consuming. This is why MALDI Mass Spectrometry approach has been developed and is now able to determine the species within a few minutes. The first approach, based on bacterial mass fingerprinting, is now well known and widespread in clinical microbiology, but it requires commercial software package. We decide here to explore an alternative approach based on proteomics and using genomic databases freely available online. One of our objectives was to develop an automated method performing high throughput identification. MALDI MS/MS experiments were performed after [i]in situ[/i] trypsinolysis, directly on MALDI plate. For this approach we used the on line UniProt database (http:/www.uniprot.org). Samples, after trypsin digestion, were analyzed in parallel with nano LC-ESI-MS/MS technique as a control. All mass spectra were recorded using a hybrid quadrupole time-of-flight mass spectrometer QStar XL. We developed the method for five bacterial genera: [i]Lactobacillus, Streptococcus, Lactococcus, Enterococcus[/i], and [i]Propionibacterium[/i]. A total of 26 species of food interest, represented by type strains, were then studied. [i]Staphylococcus aureus [/i](type strain) was also analyzed in order to have a point of comparison with a species extensively sequenced for which a very large number of proteomic data exists in databases. For a species, the higher number of strains sequenced, the more efficient the identification by proteomic approach. Thus, the limit of our method is the number of data available for each species in database. Thanks to the new generation sequencing technologies, the number of bacterial species sequenced is increasing exponentially and this would not be a limit any longer. Thus, the obtained results highlight the interest of the proteomic approach for identification by Mass Spectrometry

Proteomic approach for bacteria of food interest identification: on-plate trypsinolysis followed by MALDI-MS/MS

Whatever the field (clinical or environmental), classification and identification of bacterial strains is fundamental. For fermented foods industries, bacterial identification is a key element for risk assessment. Bacteria of food interest, mainly represented by lactic acid bacteria, are a major part of many fermented products and are found in a variety of environments. Thus the development of a rapid and simple method for their identification is particularly relevant. Most of the techniques currently used are based on molecular approaches or conventional carbohydrates use tests. However, these methods are generally time consuming. This is why MALDI Mass Spectrometry approach has been developed and is now able to determine the species within a few minutes. The first approach, based on bacterial mass fingerprinting, is now well known and widespread in clinical microbiology, but it requires commercial software package. We decide here to explore an alternative approach based on proteomics and using genomic databases freely available online. One of our objectives was to develop an automated method performing high throughput identification. MALDI MS/MS experiments were performed after [i]in situ[/i] trypsinolysis, directly on MALDI plate. For this approach we used the on line UniProt database (http:/www.uniprot.org). Samples, after trypsin digestion, were analyzed in parallel with nano LC-ESI-MS/MS technique as a control. All mass spectra were recorded using a hybrid quadrupole time-of-flight mass spectrometer QStar XL. We developed the method for five bacterial genera: [i]Lactobacillus, Streptococcus, Lactococcus, Enterococcus[/i], and [i]Propionibacterium[/i]. A total of 26 species of food interest, represented by type strains, were then studied. [i]Staphylococcus aureus [/i](type strain) was also analyzed in order to have a point of comparison with a species extensively sequenced for which a very large number of proteomic data exists in databases. For a species, the higher number of strains sequenced, the more efficient the identification by proteomic approach. Thus, the limit of our method is the number of data available for each species in database. Thanks to the new generation sequencing technologies, the number of bacterial species sequenced is increasing exponentially and this would not be a limit any longer. Thus, the obtained results highlight the interest of the proteomic approach for identification by Mass Spectrometry