Browsing by Subject "P. freudenreichii"

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  • Sillanpää, Annika (Helsingin yliopisto, 2020)
    The scientific data has demonstrated that surface-associated proteins have a significant role affecting the adaptation to GIT environment, adherence to the intestinal mucus and other potential health benefits occurring through cross-talk of propionibacteria and the host. The reported achievements on the complementary proteomic approaches optimize the accuracy of surface protein identification and surface proteins related to anti-inflammatory activities and adhesion has been identified from Propionibacterium freudenreichii strains. Thus, the aim of this thesis was to compare the effect of atmospheric conditions on surfaceome expression of the P. freudenreichii type strain DSM 20271. Bacterial cultures cultivated in aerobic and anaerobic atmosphere were harvested at the mid-exponential phase of growth. Samples were subjected to gel-free proteomic analysis, based on direct analysis of peptides acquired by trypsin cell-surface shaving followed by identification of released surface-attached proteins and peptides using LC-MS/MS and label-free quantification. It was demonstrated in this work that different atmospheric conditions highly influenced the protein expression patterns. Overall, the expression of more than a hundred proteins were affected by the change of environmental condition, of which the majority were predicted not to include either classical nor non-classical secretion motifs. It is still unresolved question if these cytoplasmic proteins in the shaved-fraction could be transported to the bacterial surface area by uncharacterized mechanisms to serve a specific moonlighting function. Few of the identified proteins, most of which were up-regulated in aerobic growth condition, were already described in other bacterial species to be involved with general stress response (ClpP, ClpB, and Ctc) and reduction of reactive oxygen species (Tpx, AhpC, and AhpF).
  • Niemi, Justiina (Helsingin yliopisto, 2020)
    Propionibacterium freudenreichii is a dairy propionic acid bacterium and it is used as adjunct culture in Swiss-type cheese manufacture. The bacterial strain is known for its GRAS (Generally Recognized as Safe) status, and it is safe to use in food. Based on previous studies, P. freudenreichii requires anaerobic conditions to start bacterial growth. Bacterial viruses ‘bacteriophages’ uses two life cycles, lysogenic or lytic cycle. The life cycle of bacteriophages affects bacterial cell lysis or division. Researchers are interested in fortifying foods naturally with vitamin B12. P. freudenreichii is known for its ability to actively produce vitamin B12. This master's thesis aims were to study 1) The effect of oxygen contentration on Propionibacterium freudenreichii subsp. freudenreichii JS7 strain and 2) the effect of oxygen consentration on the prevalence of bacteriophage in P. freudenreichii JS7 strain. In this study, P. freudenreichii JS7 strain was grown at different oxygen concentrations. Bacterial growth was observed by cell density measurements and cell plating. In the preliminary experiment, the strain was grown 1) anaerobically 2) two stages which includes aerobic and anaerobic growth conditions and 3) aerobically. Based on the partial results of the preliminary experiment, the fermentation was performed in a bioreactor in an anaerobic growth condition. The cell plating results of the bioreactor experiment were used to study the effect of oxygen concentration on the presence of bacteriophage in a bacterial strain by using three primer pairs. The polymerase chain reaction was applied to study the presence of the bacteriophage genome as a plasmid, free, and prophage which means that phage genome is integrated into the bacterial chromosome. Based on the results of this study, the cell density of P. freudenreichii JS7 strain increases faster in anaerobic oxygen concentration comparing to aerobic and two staged oxygen concentrations. Based on the results of the bioreactor study, the bacterium can anaerobically form different size of colonies for the cell plating. The bacteriophage genome may be present as a plasmid, free and integrated into the bacterial chromosome. The results of the bacteriophage study might give signs that P. freudenreichii JS7 could be able to utilize the lysogenic life cycle in anaerobic growth condition.
  • Ververis, Ermolaos (Helsingfors universitet, 2016)
    Cobalamin (vitamin B12) occurs naturally in some animal-derived foods and is produced exclusively by microorganisms. An optimised protocol was used for extraction of cobalamin from cheese matrixes. No pseudocobalamin was detected in any of the examined samples. Cobalamin levels (mg/100g FW) detected in commercial emmental cheeses of three ripening stages did not alter significantly (P>0.05). Similar results were observed during the ripening of experimental semi-hard cheeses with or without propionibacteria. Existence of propionibacteria as adjunct culture in experimental cheeses did not alter significantly contribution on cobalamin levels of the cheese (P>0.05). The findings indicate that in the studied cheese matrixes the presence of propionibacteria did not affect the amount of cobalamin. The conditions to which propionibacteria are subjected during cheese manufacture and ripening and the presence of adenosyl-cobalamin in milk may be factors that repress cobalamin synthesis in Swiss- type cheeses. To date, the only known food grade microorganism that can produce cobalamin is Propionibacterium freudenreichii. This microorganism can also produce small amounts of pseudocobalamin, a compound structurally similar to cobalamin. BluB/CobT2 fusion gene is the factor that differentiates the two compounds upon their biosynthesis, by synthesizing and binding 5,6-dimethylbenzimidazole (DMBI) to the final molecule of cobalamin. In the present study, attempts to inactivate this gene were performed in order to investigate the existence of an alternative enzyme, capable of activating adenine for attachment as a lower ligand in pseudocobalamin, instead of DMBI. An electroporation protocol was implemented in order to transform plasmids containing bluB or cobT2 fragments and gene encoding erythromycin resistance in propionibacteria. Following transformation plasmid carrying bacteria were selected by cultivation in medium containing erythromycin. Homologous recombination of the bacterial genome and the non-replicative plasmid was expected to occur, leading to insertional mutagenesis. Colonies appeared after 7 and 11 days and were identified as propionibacteria but the disruption of bluB/cobT2 gene could not be verified. Inefficient transformation protocol, satellite colonies, low transformation efficiency, or choice essentiality of the bluB/cobT2 are among the possible explanations for the outcome of the experiment. Electroporation conditions should be optimized towards a more efficient P. freudenreichii transformation.