Browsing by Subject "vitamin B12"

Microalgae are promising raw materials for food- and biotechnology because they contain a lot of proteins, unsaturated fatty acids, pigments, vitamins and minerals. There are few studies on vitamin B in microalgae and some of them are based on partly inaccurate methods. Microalgae in general, analytical methods regarding their analysis and how they use vitamins were discussed in the literature part of this thesis. The structures, chemical properties and occurrence in foods as well as commonly used analytical methods of the vitamins in question were presented. The aim of the experimental part of this thesis was to analyse commercially marketed microalgae supplements (Chlorella sp. and Arthrospira sp. (spirulina)) and laboratory-grown microalga (Euglena gracilis) as potential sources of folate, niacin, vitamin B2 and B12. Contents of vitamin B12, B2 and niacin were analysed using UHPLC method separately validated for each vitamin. The total folate content was analysed microbiologically and folate vitamers by using UHPLC. The vitamin B12 was analysed microbiologically and the active forms of vitamin B12 were confirmed using LC-MS. Acid hydrolysis was used in analysing niacin content. The total folate content in chlorella supplements was of the same order when analysed microbiologically or with UHPLC. Instead, in spirulina supplements the microbiologically analysed total folate content was higher than the total folate content based on the sum of folate vitamers analysed with UHPLC. At most, the total folate content of E. gracilis -sample was 3-fold higher than in commercial microalgae supplements. Especially in spirulina supplements, the vitamin B12 contents were clearly higher when analysed microbiologically than they were when analysed with UHPLC. The difference was most likely due to pseudocobalamin that resembled vitamin B12. On average E. gracilis -samples had higher vitamin B2 content than the commercial supplements. E. gracilis -samples and chlorella supplements contained more niacin than spirulina supplements. According to this thesis, commercially marketed microalgae supplements contained different amounts of vitamin B. Chlorella sp. was proved to be a great source of folate, vitamin B12 and niacin and moderate source of B2. The majority of vitamin B12 in Arthrospira sp. (spirulina) was pseudocobalamin. Despite that, spirulina supplements proved to be a moderate source of vitamin B12. On average, E. gracilis had the highest vitamin B content and it would potentially be an excellent source of vitamin B – if it was accepted for food use.

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.

Legume consumption and cultivation is recommended due to several health and environmental benefits. However, legumes naturally lack vitamin B12 and contain non-digestible, fermentable raffinose family oligosaccharides (RFOs) which are found to cause abdominal symptoms. Propionibacterium freudenreichii was earlier found to produce significant B12 contents into plant-based matrices by fermentation. Moreover, many of lactic acid bacteria used in food fermentation are found to produce the enzyme α-galactosidase which degrades the RFO compounds. This study aimed investigating the use of fermentation for a simultaneous B12 production and RFO reduction. The study examined direct fermentation with P. freudenreichii and co-fermentation with starters. In relation to B12, the study examined if B12 content and RFO reduction positively correlated after the fermentation process. The state of RFO breakdown was explored by detecting the RFO levels with high performance anion exchange chromatography combined with a pulse amperometric detector (HPAEC-PAD) after every 24 h during the 72 h of fermentation. Several fermentation schemes showed either efficient or complete RFO degradation and produced active B12 at significant level with some minor differences between them. Generally, direct fermentation led to superior RFO reduction compared to co-fermentation in the studied matrices. The study concluded the inversely proportional relation between RFO reduction and B12 formation when co-fermented with a commercial starter.