Browsing by Subject "cobalamin"

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  • Deptula, Paulina; Chamlagain, Bhawani; Edelmann, Minnamari; Sangsuwan, Panchanit; Nyman, Tuula A.; Savijoki, Kirsi; Piironen, Vieno; Varmanen, Pekka (2017)
    Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein andmetabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.
  • Mercier, Léon (Helsingin yliopisto, 2018)
    PURPOSE AND GOALS Microalgae are unicellular eukaryotic organisms capable of photosynthesis. They harvest sunlight and efficiently take up carbon dioxide and nutrients such as nitrogen and phosphorus from their environment and use them for their growth. Due to these properties, their rapid growth and ability to survive in a variety of environments, microalgae have potential in biotechnological applications that promote nutrient recovery and recycling, water purification and the carbon neutral production of biochemicals and possibly biofuels. The purpose of this study was to investigate the suitability of a side stream water originating from the production of baker’s yeast (yeastwater) for the cultivation of a species of microalga called Euglena gracilis. The study aimed to determine the capacity of this water to support growth and protein production of E. gracilis as well as the capacity of E. gracilis to remove nutrients from the water. The effect of filtration of the water on these parameters was also studied. Yeastwater contains an organic molecule called betaine in relatively high concentrations. Betaine has previously been shown to boost the production of the important vitamin cobalamin in bacteria. The study aimed to determine the effect of betaine on the growth of E. gracilis and on the production of cobalamin in the algal-bacterial symbiosis. METHODS E. gracilis was cultured in laboratory scale photobioreactors. Its growth, protein production and nutrient uptake capacity was determined. Baker’s yeast production side stream water diluted with MQ-water was used as the growth medium either in filtered or unfiltered form. A control treatment was prepared where no microalgal inoculate was added to the photobioreactor. The same microalga was also grown in a synthetic nutrient medium with and without betaine. The uptake of betaine and biomass concentrations of cobalamin were determined. For the determination of microalgal growth, dry weight determination and flow cytometry analysis were used. Protein production was determined on the basis of total nitrogen concentration in the biomass. Spectrophotometric measuring kits were used for the determination of nutrient concentrations. Liquid chromatography techniques were used for the determination of betaine and cobalamin concentrations. RESULTS Significant microalgal growth was observed in filtered yeastwater, while growth in unfiltered yeastwater was very low. Nitrogen removal was higher in presence of E. gracilis compared to the control treatment. Protein production in yeastwater was comparable to that of microalgae grown in synthetic medium. E. gracilis grew much better in the synthetic media supplemented with betaine than without the addition. Betaine enrichment had no effect on cobalamin production. Cobalamin was produced in unfiltered yeastwater both with and without the presence of E. gracilis. CONCLUSIONS Unfiltered yeastwater does not support growth of E. gracilis possibly due to its high turbidity. Filtered yeastwater, on the other hand can support the production of E. gracilis biomass. E. gracilis can be used to reduce nitrogen concentrations in yeastwater. Yeastwater can support cobalamin production by bacteria, but this phenomenon did not benefit from the presence of the microalga. The effect of betaine on microalgal growth warrants further study to determine whether it is related to the accumulation of intracellular nutrients, storage compounds or to some other phenomenon. Yeastwater is a promising nutrient feedstock for microalgal biomass production. However, the role of filtration and possibility of using other methods for turbidity reduction needs to be further studied.
  • 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.
  • Ishchenko, Tatiana (Helsingfors universitet, 2014)
    Propionibacterium freudenreichii is commonly known as a Swiss-type cheese ripening culture, and is also frequently applied in the industrial production of vitamin B12 (cobalamin). One way to increase vitamin B12 productivity is the supplementation of medium with 5,6-dimethylbenzimidazole (DMBI), the lower ligand of cobalamin. The aim of this thesis was to investigate the effect of exogenous DMBI on vitamin B12 synthesis and protein production in type strain of P. freudenreichii ssp. freudenreichii. Results showed that DMBI supplementation improved cobalamin production 2.7- and 2.0-fold in cultures grown in anaerobic and two-step processes, respectively. Two-step incubation enabled the achievement of significantly higher levels of cobalamin both in the absence and presence of DMBI. The highest cobalamin level of 0.6 ?g/ml was achieved by the combination of DMBI supplementation with the two-step process. Proteome maps displayed 474 protein spots which is less than 20% of P. freudenreichii predicted proteome. No significant differences in protein production were found between cultures grown in the absence and presence of DMBI. Only two protein spots were more abundant in presence of DMBI and four spots showed higher abundance in absence of DMBI. None of detected proteins from these spots were found to be directly involved in cobalamin biosynthesis.