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

The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications

Background: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. 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 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings: The 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/Significance: With 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

Variability of Bacterial Biofilms of the “Tina” Wood Vats Used in the Ragusano Cheese-Making Process▿

Ragusano cheese is a “protected denomination of origin” cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called “tinas” were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system

Microbiote des produits fermentés par approches culturales et métagénomiques Exemple d'un fromage artisanal : le Pélardon

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

Measurement as an ontological scalpel

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