In addition to their use in cheese technology, dairy propionibacteria have been identified as potential probiotics. However, to have a probiotic effect, propionibacteria have to survive and to remain metabolically active in the digestive tract. The aim of the present study was to investigate the survival and metabolic activity of Propionibacterium freudenreichii within the gastrointestinal tract of human microbiota-associated rats, and its influence on intestinal microbiota composition and metabolism. Twenty-five dairy Propionibacterium strains were screened for their tolerance towards digestive stresses and their ability to produce propionate in a medium mimicking the content of the human colon. Three strains were selected and a daily dose of 2 x 10(10) colony-forming units was fed to groups of human microbiota-associated rats for 20 d before microbiological, biochemical and molecular investigations being carried out. These strains all reached 8-log values per g faeces, showing their ability to survive in the gastrointestinal tract. Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. The probiotic efficacy of propionibacteria was yet species- and strain-dependent. Indeed, two of the strains, namely TL133 and TL1348, altered the faecal microbiota composition, TL133 also increasing the caecal concentration of acetate, propionate and butyrate, while the third strain, TL3, did not have similar effects. Such alterations may have an impact on gut health and will thus be taken into consideration for further in vivo investigations on probiotic potentialities of P. freudenreichii.

Dairy propionibacteria are present in Graviera Kritis, a traditional Gruyère-type cheese made without added propionic starter. Ten isolated strains were identified by a combination of SDS-PAGE, species-specific PCR and according to their ability to ferment lactose. They were all found to belong to the Propionibacterium freudenreichii subsp. shermanii species. Because of the stressing Gruyère technology, which includes cooking at 52 to 53 °C their thermotolerance was investigated at 55 °C. Thermotolerant and thermosensitive strains were clearly discriminated. Interestingly, the reference strain CIP 103027 belongs to the sensitive subset. One sensitive strain, ACA-DC 1305 and one tolerant, ACA-DC 1451, were selected for further study and compared to CIP 103027. For the sensitive strains ACA-DC 1305 and CIP 103027, heat pre-treatment at 42 °C conferred thermoprotection of cells at the lethal temperature of 55 °C, while there was less effect on the tolerant ACA-DC 1451. No cross-protection of salt-adapted cells against heat stress was observed for none of the strains. Differential proteomic analysis revealed distinct but overlapping cell responses to heat stress between sensitive and tolerant strains. Thermal adaptation upregulated typical HSPs involved in protein repair or turnover in the sensitive one. In the tolerant one, a distinct subset of proteins was overexpressed, whatever the temperature used, in addition to HSPs. This included enzymes involved in propionic fermentation, amino acid metabolism, oxidative stress remediation and nucleotide phosphorylation. These results bring new insights into thermoprotection in propionibacteria and the occurrence of divergent phenotypes within a same subspecies.

This study investigated Propionibacterium freudenreichii tolerance to stresses encountered during food technology or in the digestive tract. The impact of food matrices on stress survival was evaluated. The ability of P. freudenreichii strains SI 41 and CNRZ 81 to survive both acid and bile salts stresses was studied in vitro. Stress tolerance was examined after inclusion of bacteria in different food matrices (alginate beads, xanthan–gellan beads, fermented milk) and compared to the stress tolerance of the same strains in pure cultures (either in exponential growth phase, in stationary phase, or after freeze-drying). Fermented milk was determined as the best probiotic vector to protect propionibacteria from this trial. This in vitro investigation gave promising results. Indeed, some food matrices can significantly improve protection of bacterial cells from stress injury. These data, which should be confirmed in an in vivo study, will be taken into consideration for the improvement of technological processes and for the choice of an adequate probiotic vector.

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.

Dairy propionibacteria, and especially Propionibacterium 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 ill 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.

ABSTRACT We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria.

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C, A prophage-cured derivative of P, freudenreichii was readily isolated and subsequently relysogenized, Lysogeny was therefore formally demonstrated in P. freudenreichii.

The probiotic application of dairy propionibacteria as well as their use in cheese technology implies exposure to various environmental stresses, including acidic pH. The acid tolerance response (ATR) of Propionibacterium freudenreichii was investigated. One strain present in Swiss-type cheese proved to be acid-tolerant, since no lethal effect was observed during exposure at pH 3. Moreover, survival at pH 2 (acid challenge) was conferred by pre-exposure to a moderate acid stress (acid adaptation). This adaptative response was triggered quickly, and showed a maximal efficiency upon exposure to pHs between 4 and 5. Stationary phase ATR and acid habituation were also demonstrated, and conferred increased survival at pH 2 without pre-exposure. Exponentially-growing bacteria were partially protected towards acidity by pre-exposure to other stresses (heat, starvation, but not hyperosmolarity). A comparative study of different strains revealed that acid stress susceptibility is strain-dependent within this species. Adaptation and survival at low pH is likely to determine the efficacy of a P. freudenreichii strain both as a cheese starter and as a probiotic.

Deux types de bacteriophages (phages) infectant des bacteries propioniques laitieres (BPL) ont ete isoles a partir de fromages a pâte pressee cuite. L'un appartient au groupe B1 de la classification decrite par Bradley alors que l'autre est a notre connaissance le premier virus filamenteux infectieux decrit a ce jour cher une bacterie a Gram positif. Les deux types de phages ont ete identifies dans plusieurs fromages a pâte pressee cuite differents. Leur origine pourrait etre le lait cru ou de faibles quantites de phage ont ete detectees. Les phages du groupe B1 peuvent etre portes par les BPL a l'etat de prophage. L'hybridation de l'ADN montre que tous les phages de BPL du groupe B1 sont etroitement lies, bien qu'ils aient ete isoles de fromages differents. Ils ont donc probablement une origine commune. La flore propionique dominante a la fin de l'affinage des fromages peut etre constituee d'une souche de BPL sensible. Il semble des lors evident que les phages n'en perturbent pas le developpement et par consequent n'ont pas d'effet nefaste sur les proprietes organoleptiques des fromages a pâte pressee cuite. Ces donnees nous ont par ailleurs permis de mettre au point un vecteur de clonage efficace chez Propionibacterium freudenreichii.

Genomic DNAs of 22 strains of Lactobacillus helveticus of various geographical origins were analyzed by pulsed-field gel electrophoresis. Two endonucleases, SmaI and SgrAI, of the 19 tested produced DNA fragments useful for strain comparison. With the endonuclease SmaI, a characteristic restriction pattern was identified for 18 of the 22 strains. The percentage of similarity (Dice coefficient) between the profiles varied between 26% and 100%, and clustering was accomplished by using the unweighted pair group method with arithmetic averages (UPGMA). For the strains showing identical profiles,the high genomic similarity was confirmed when the endonuclease SgrAI was used instead of SmaI. From summation of SmaI and SgrAI fragments from three L. helveticus strains(CNRZ 241, CNRZ 303, and CIP 57.15), the genomic length was estimated at ca.1. 85-2.0 Mb.

Restriction endonuclease patterns generated by Pulsed-Field Gel Electrophoresis (PFGE) were used to compare 96 strains of dairy propionibacteria originating from dairy products, international and industrial collections; endonucleases XbaI and SspI gave satisfactory restriction patterns. However, whereas XbaI can be used for Propionibacterium freudenreichii, SspI seems more suitable for the three other species: P. acidipropionici, P. thoenii, and P. jensenii. It is a convenient method to differentiate the dairy propionibacteria from closely related bacteria and from others usually present in dairy products. We observed a considerable restriction fragment length polymorphism among the Propionibacterium chromosomes and especially for P. freudenreichii: among 48 strains we detected 40 different patterns. This species is the most commonly encountered in the Swiss-type cheeses and is the only Propionibacterium species used as a cheese starter. Conversely, the species P. acidipropionici is not very diverse: among nine strains we observed only four different patterns, two of which were closely related. This is probably because this species is not used as a starter in cheese manufacture and consequently is poorly represented in collections. When strains come from geographical different isolates, their patterns are always different with very few common bands. The presence of numerous identical strains was due to the fact that they were present at the same time in the national collections, research laboratory collections, and in the industrial ones.

We isolated bacteriophages active against Propionibacterium freudenreichii from 16 of 32 swiss cheese samples. Bacteriophage concentrations ranged from 14 to 7 x 10(5) PFU/g, depending on the sample and the sensitive strain used for detection. Only a few strains, 8 of the 44 strains of P. freudenreichii in our collection, were sensitive. We observed that multiplication of bacteriophages occurred in the cheese loaf during multiplication of propionibacteria in a warm curing room, but it seems that these bacteriophages have no adverse effect on the development of the propionic flora. We also found that sensitive cells, originating from either the starter or the cheese-making milk, were present at a high level (10(9) CFU/g) in the cheese.

Dairy propionibacteria, especially Propionibacterium freudenreichii, are widely used in Swiss cheese manufacture because oftheirabilitytoproducethecharacteristicflavorandholes(6,

Nineteen bacteriophages infecting P freudenreichii have been isolated from 32 Swiss-type cheeses. They all present a similar morphology and belong to the B1 group of Bradley's classification. According to their restriction patterns, obtained with the endonuclease Pstl, they can be grouped into 3 clusters, 1 of which contains the majority of the phages. Sixteen of the 32 cheeses studied were infected with bacteriophages. We observed that all the cheeses made from raw milk and ripened in a warm curing room for several weeks which are known to contain high concentrations of propionibacteria, were infected with bacteriophages. Moreover, the multiplication of bacteriophages occurred in cheese during the multiplication stage of propionibacteria in a warm curing room. We have shown that raw milk contains bacteriophages and thus it can be a source of phages in facto:ies. However, these bacteriophages are sensitive to the milk heat treatment used for cheesemaking.

Retention behaviour of two lactococcal phages added to raw milk submitted to cross flow microfiltration with a 0.1 mu m pore size membrane was studied. The microfiltrate, 'milk without casein', contains 0.4-0.14% of the phage particles added to the milk whatever the phage type and the level of inoculation. Such a retention cannot be explained by electrostatic interactions between phage particle and casein micelle or microfiltration membrane but is more probably due to a reduction of membrane pore size resulting from a concentration polarization phenomenon.

We examined 70 dairy propionibacteria and detected a crystalline surface layer (S-layer) in only 2 organisms (Propionibacterium freudenreichii CNRZ 722 and Propionibacterium jensenii CNRZ 87) by freeze-etching and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). Both S-layers exhibited oblique (p2) symmetry (a = 9.9 nm; b = 5.4 nm; gamma = 80 degrees) and completely covered the cell surface. Treatment for 15 min at the ambient temperature with 5 M guanidine hydrochloride or acidic conditions (250 mM ammonium acetate, pH 2.7) efficiently extracted the S-layer protein from intact cells of strain CNRZ 722, whereas treatment with 5 M guanidine hydrochloride at 100 degrees C for 15 min was necessary to isolate the S-layer protein of strain CNRZ 87. The precipitates obtained after dialysis of the extracting agents produced no regular patterns. The molecular masses of the two S-layer proteins, as estimated by SDS-PAGE, were 58.5 kDa for the strain CNRZ 722 and 67.3 kDa for the strain CNRZ 87. Mass spectrometry of the isolated S-layer protein of strain CNRZ 722 gave a molecular mass value close to the expected value (56,533 Da). The N-terminal sequences of the two purified S-layer proteins differed, as did their amino acid compositions, except that the same high hydrophobic amino acid content (52%) was observed.

Bacteriophage 822 was isolated from a Swiss-type cheese. Out of 30 strains of Propionibacterium freudenieichii, only the strain TL 110 was sensitive to this phage. 822 morphology is very similar to those of phages infecting P acnes. It has an' isometric head, a non-contractile tail and a tail plate, so it belongs to the 81 group of 8radley's classifiCation. The 822 genome consists of a Iinear double-stranded DNA molecule 40 kb long with cohesive ends. .