Browsing by Subject "B12-vitamiini"

B12-vitamiini on rakenteeltaan monimutkainen, vesiliukoinen kompleksiyhdiste. Ihmisille aktiivisessa B12-vitamiinin muodossa alempana ligandina on 5,6-dimetyylibentsimidatsoli. B12-vitamiinia saadaan eläinperäisistä elintarvikkeista. Tämän tutkielman kirjallisuusosio käsitteli B12-vitamiinin rakennetta, vitamiiniaktiivisuutta, ruokavalion vaikutusta saantiin ja imeytymisen vaiheita ihmisen elimistössä. Sen lisäksi käsiteltiin B12-vitamiinin analytiikkaa ja pitoisuuksia elintarvikkeissa. Useissa tutkimuksissa B12-vitamiinia on määritetty mikrobiologisella menetelmällä. Menetelmän epäillään nykyisin vääristävän B12-vitamiinipitoisuuksia, koska menetelmässä käytetty testiorganismi reagoi positiivisesti myös B12-vitamiinin kaltaisiin muihin korrinoidiyhdisteisiin. Tämän tutkielman kokeellisen osion tavoitteena oli tutkia kuinka paljon eläinperäiset elintarvikkeet sisältävät B12-vitamiinia. Tutkimukseen valittiin keskeisiä eläinperäisiä elintarvikkeita sekä kaksi elintarvikkeeksi hyväksyttyä hyönteislajia. Tavoitteena oli myös verrata toisiinsa mikrobiologista ja erittäin suuren erotuskyvyn nestekromatografista (UHPLC) menetelmää B12-vitamiinin määrityksessä sekä tutkia eri uuttomenetelmien vaikutusta analysoituihin B12-vitamiinipitoisuuksiin. Uuttokokeiden tulosten perusteella kaikille näytteille ei voitu käyttää samaa esikäsittelymenetelmää. Uutto pepsiinikäsittelyllä paransi määritettyä B12-vitamiinipitoisuutta kirjolohella, keltuaisella, naudan sisäpaistilla ja maidolla, kun taas pankreatiinikäsittely paransi määritettyä B12-vitamiinipitoisuutta juustoilla ja silakalla. Elintarvikkeista odotetusti eniten B12-vitamiinia oli naudan ja porsaan maksan lisäksi silakassa, juustoissa ja naudan sisäpaistissa. Vähiten B12-vitamiinia oli broilerin rintafileessä. Mikrobiologisella ja UHPLC-menetelmällä määritetyissä B12-vitamiinipitoisuuksissa oli merkittävä ero menetelmien välillä. Liha- ja kalanäytteiden B12-vitamiinipitoisuudet olivat 7–64 % pienempiä UHPLC-menetelmällä kuin mikrobiologisella menetelmällä määritettynä, maitotuotteilla ja keltuaisella 20–67 % pienempiä ja myös hyönteisillä eroa oli 71–81 %. Mikrobiologinen menetelmä on herkkä ja edullinen käyttää. Jatkossa elintarvikkeiden B12-vitamiinipitoisuus kannattaa kuitenkin määrittää UHPLC-menetelmällä, joka erottaa ihmiselle aktiivisen B12-vitamiinin muodon muista ei-aktiivisista muodoista eikä anna vastetta muille B12-vitamiinin kaltaisille yhdisteille.

The usual dietary sources of vitamin B12 are animal-based foods such as meat, milk, fish and shellfish. Vitamin B12 is an essential water-soluble vitamin for humans, which have many necessary tasks in human body. People who lack animal foods like vegetarians and vegans might be at risk of vitamin B12 deficiency. Microorganisms are the only original sources of vitamin B12 in nature. Especially Propionibacterium freudenreichii synthesizes active form of vitamin B12 for humans. P. freudenreichii is GRAS -graded bacteria and it is safe to use in food matrices. Recently, it has been used for natural fortification of vitamin B12 in plant-based products by fermentation. However, the bioaccessibility of vitamin B12 of those products in human body is not very well known. In the beginning of the study, stability of different B12 vitamers were studied in different light and pH conditions. Vitamin B12 forms are very light-sensitive, especially its physiological forms methyl- and adenosylcobalamin. The aim of this research was to study bioaccessibility of vitamin B12 from P. freudenreichii cells and selected food products using a static in vitro assay. After the in vitro model, vitamin B12 content was analysed with UHPLC system. In addition of bacterial cell samples, bioaccessibility was studied also in some food samples, like bread, pasta and spray-dried powder, fortified with P. freudenreichii cells. In vitamin B12 stability studies, red light seemed to improve the stability of B12 forms. In yellow light methyl- and adenosylcobalamin degraded after 15 minutes. In red light they were detectable after hours of exposure. Methylcobalamin seems to be the most sensitive form of vitamin B12 vitamers. The study revealed that the bioaccessibility of B12 was very small (1,5 %) in P. freudenreichii cells but over 50% in whole bacterial broth. Heat treatment for the samples improved the bioaccessibility to some extent. In B12 fortified food samples, the bioaccessibility of B12 was very good (>70 %). The number of heat treatments and food structure could be one reason why bioaccessibilities in food samples are better than in cell samples. According to this research, in situ fortified food products could be a promising source of vitamin B12 in future.

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.

Propionibacterium freudenreichii is a known as secondary ripening culture that occurs spontaneously in small amounts in raw milk. Contamination of raw milk with P. freudenreichii and other microbes can occur during milking, transporting and processing, either from the air or through surfaces. Microbes that spontaneously end up in raw milk survive on heat treatments that are milder than pasteurization, which makes the microbiome of raw milk cheeses more diverse than cheeses made from pasteurized milk, where a higher heating temperature inactivates enzymes and kills microbes. During the ripening of the cheese, a lot of changes occur in the microbiome. The aim of this master thesis was to discover the presence and number of lactic acid bacteria and propionic bacteria in raw milk cheeses by culturing samples in different culture media, and to identify P. freudenreichii by using a polymerase chain reaction (PCR) method and P. freudenreichii -specific primers. In addition, selected P. freudenreichii strains were tested for their ability to produce vitamin B12 by liquid chromatography and the effects of different NaCl concentrations on the growth of P. freudenreichii isolates were investigated. There were differences in the number bacterial colony forming units of the cheeses. Of the selected catalase-positive isolates resembling P. freudenreichii isolates morphologically, 65% were identified as P. freudenreichii strains by PCR. Most strains of P. freudenreichii were identified from isolates of Vorarlberger Bergkäs and Liechtensteiner cheese samples (92–94%). The least strains of P. freudenreichii were identified from isolates of Tete de Moine cheese samples (25%). Possibly Vorarlberger Bergkäs and Liechtensteiner cheeses had more P. freudenreichii bacteria compared to other propionic bacteria with similary colony morphology, and Tete de Moine had less of P. freudenreichii bacteria than other propionic bacteria. Based on growth experiments, increasing the NaCl content on agar plates slowed the growth of P. freudenreichii strains and impaired the color of the colonies. Based on the vitamin assays, all 14 strains of P. freudenreichii tested produced active vitamin B12 in humans, but there were differences in the amounts of vitamins produced per strain. Strain-specific differences were also observed in the ratios of vitamin forms produced (vitamin B12 / pseudovitamin).

Tausta ja tavoitteet: Punaisen lihan kulutus on maailmanlaajuisesti liian runsasta, millä on merkittäviä epäsuotuisia ympäristö- ja terveysvaikutuksia. Suomessa erityisesti miehet syövät merkittävästi enemmän punaista lihaa kuin ravitsemussuosituksissa suositellaan. Punainen liha on kuitenkin suomalaisten miesten ruokavalioissa merkittävä B12-vitamiinin ja hyvin imeytyvän raudan lähde. Työn tavoitteena oli tutkia punaisen ja prosessoidun lihan osittaisen korvaamisen palkokasveilla vaikutuksia suomalaisten miesten B12-vitamiinin ja raudan saantiin sekä niiden biomarkkereihin. Menetelmät: Tutkimusjaksoa edeltävän seulonnan läpäisyn jälkeen osallistujat (n=102) jaettiin kahteen rinnakkaisryhmään, liharyhmään ja palkokasviryhmään. Liharyhmä sisällytti ruokavalioonsa 760 grammaa punaista lihaa viikossa, kun palkokasviryhmä puolestaan sisällytti ruokavalioonsa 200 grammaa punaista lihaa ja loput punaisesta lihasta (560 g/vk) saatavasta proteiinimäärästä korvattiin palkokasveja sisältävillä elintarvikkeilla. Muu ruokavalio pyydettiin pitämään tavanomaisena, mutta muiden kuin tutkimuksessa jaettujen punaisen lihan ja palkokasvien käyttö kiellettiin. Tulokset: B12 vitamiinin saanti oli suurempaa (p<0,001) liharyhmässä (7,7±4,0 μg/vrk) kuin palkokasviryhmässä (5,0±2,7 μg/vrk). Raudan saanti oli puolestaan pienempää (p<0,001) liharyhmässä (14,0±3,1 mg/vrk) kuin palkokasviryhmässä (21,9±5,7 mg/vrk). Transkobalamiini II sitoutuneen B12-vitamiinin (holoTC) pitoisuus oli suurempi (p=0,022) liharyhmässä (120,4±47,3 pmol/l) kuin palkokasviryhmässä (107,1±45,1 pmol/l). Interventioryhmien välillä ei havaittu eroja veren hemoglobiinissa ja hematokriitissa sekä plasman transferriinireseptorissa, raudassa, ferritiinissä, transferriinissä eikä transferriinin rautakyllästeisyydessä. Johtopäätökset: Punaisen lihan osittainen korvaaminen palkokasveilla vähensi B12-vitamiinin saantia ja johti pienempään veren holoTC-pitoisuuteen. Raudan saanti puolestaan kasvoi palkokasviryhmässä mutta raudan biomarkkereissa ei ollut eroja interventioryhmien välillä.

The literature review of this thesis concentrated on vitamin B12 (Clb), its forms, vitamin B12 anologues, and the biosynthetic route of Clb. In addition, the roles of ribolfavin (RF) and niacin (NAM) in this biosynthetic route were discussed. Cereals were also evaluated as a matrix for vitamin B12 synthesis based on their RF and NAM concentrations. The aims of the experimental part were to prepare a malt extract medium, to study the effects of RF and NAM, and to compare three Propionibacterium freudenreichii strains. The medium (pH 6.40) consisted of 10% of malt extract (ME) and 0.1 M potassium phos-phate buffer. Sterile filtered precursor solutions (e.g. CoCl2) were added to the autoclaved broth. The final composition of the broth was decided based on a preliminary test, where lactate (L; 8 g/L) and/or tryptone (T; 5 g/L) supplements were compared. Thus, the impact of RF and NAM to Clb yield was studied in ME+L+T medium. In these tests five conditions were used: ME+L+T, ME+L+T with DMBI (5,6-dimethylbenzimidazole), and either 1) RF levels of 1, 3, and 38 ?M (27 mM of NAM) or 2) NAM levels of 0.1, 0.6, and 27 mM (3 ?M of RF). The RF concentrations were also tested with strain 3 in ME+T broth. Strains were incubated anaerobically at 30 °C for 3 days and microaerobically for 4 days. Optical densities, cell masses, and pH values were measured. Intracellular Clb was extracted as cy-ano-Clb and quantified using an UHPLC/UV method. From medium RF and niacin were analyzed with an UHPLC/FL method, and sugars and acids with an HPLC/RI/UV method. In ME+L+T strain 1 produced 1.0 ± 0.2 ?g/mL of vitamin B12, strain 2 synthesized 1.2 ± 0.2 ?g/mL, and strain 3 yielded 0.82 ± 0.2 ?g/mL of Clb. DMBI increased Clb synthesis most in strains 1 and 3, while with strain 2 the 27 mM NAM level together with RF resulted in the highest yields. Alone low NAM concentrations did not affect Clb yields, but RF increased Clb production by strains 2 and 3 (p < 0.05). On the other hand, high RF concentration may have inhibited its intake. Thus, RF levels in cereals should be well suited for Clb synthesis. However, strains 1 and 2 had higher Clb yields and they salvaged RF more than strain 3. Furthermore, the results indicated that with these two strains NAM may stimulate Clb synthesis or growth. However, these results should be confirmed. Moreover, further studies are needed especially on the role of NAM and nicotinic acid, the salvage routes of niacin and RF, and other nutritional requirement of the strains in cereal matrices.