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Browsing by Subject "B12-vitamiini"

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  • Lehtonen, Heidi (2018)
    Vitamin B12 is a water-soluble molecule with a complex structure. In the active form to humans B12 has to have 5,6-dimethylbenzimidazole as a lower ligand. B12 is synthesised only by certain bacteria and natural sources in the human diet are restricted mainly to foods of animal origin. The exact structure of B12 and vitamin activity, the supply from different diets and absorption in the body were discussed in the literature part of this thesis. Also B12 determination methods and B12 levels in foods of animal origin were discussed. Usually vitamin B12 contents in foods have been obtained with a microbiological method (MBA). Currently it has been of concern that MBA may overestimate results because test organism in the MBA method reacts also to compounds similar to B12. The aim of the experimental part of this thesis was to investigate content of vitamin B12 in foods of animal origin. Further, two determination methods, MBA and ultra-high performance liquid chromatography (UHPLC), were compared with each other. The aim was also to examine the effect of different extraction methods on the yield of vitamin B12. The samples chosen to this study were beef, pork meat, chicken meat, beef and pork liver, rainbow trout, Baltic herring, egg, skim milk, yogurt and Edam and Emmental cheese. Also two insects, cricket and mealworm, were chosen to this study. The extraction tests showed that it was not possible to use one extraction method to all samples. Extraction with pepsin improved the yield of vitamin B12 in rainbow trout, egg yolk, beef and milk whereas pancreatin improved the yield in cheeses and Baltic herring. As expected B12 content was the highest in livers of beef and pork. Also beef, Baltic herring and cheeses had high concentrations of B12. Chicken meat contained the lowest concentration of B12. Comparison between the MBA and the UHPLC method proved that with MBA the vitamin B12 concentrations were much higher than with UHPLC. B12 concentrations with UHPLC were 7–64% lower in meat and fish samples. Milk products and egg yolk had 20–67% lower B12 concentrations with UHPLC and insects had 71–81% lower concentrations. MBA method is sensitive and has low reagent costs but in the future UHPLC method should be chosen for B12 analysis because it can separate the active B12 form from the inactive forms.
  • Peltonen, Liisa (2020)
    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.
  • Karanko, Ira (2023)
    Tämän tutkielman tavoitteena oli tuottaa Propionibacterium freduenreichii -bakteerin ja Rhizopus -homesienen yhteisfermentaatiolla B12-vitamiinia härkäpavusta valmistettuun tempeen. Samalla tutkittiin, mitä aminohappoja ja orgaanisia happoja tempeissä vapautui fermentaation aikana ja vaikuttivatko ne B12-vitamiinin tuottoon. B12-vitamiinia sisältävien kasviperäisten elintarvikkeiden tuottamisen tutkiminen on tärkeää, sillä se helpottaisi vegaanista ruokavaliota noudattavien mahdollisuuksia saada B12-vitamiinia ravinnostaan. Hypoteesina oli, että B12-vitamiinia saataisiin härkäpaputempeihin, mutta oletettavasti sen pitoisuuteen valmiissa tempeissä vaikuttaisi käytetty propionibakteerikanta. Ensin kasvatettiin fermentointiin käytetyt propionibakteerikannat ja valmistettiin tempenäytteet härkäpavusta. Valmiista tempeistä määritettiin pH, kosteuspitoisuus, B12-vitamiini, vapaat aminohapot, orgaaniset hapot ja ammoniakki sekä propionibakteerien ja enterobakteerien määrät. Neljästä propionibakteerikannasta kaksi tuottivat tempeen huomattavia määriä B12-vitamiinia (1 µg/g kuivapainoa kohti). Fermentaation aikana vapaiden aminohappojen pitoisuus lisääntyi kaikilla kannoilla valmistetuissa tempeissä selvästi. Eri aminohappoja vapautui kuitenkin eri kannoilla hyvin eri määriä. Maitohappo kului näytteistä fermentoinnin aikana. Pelkällä Rhizopus -homesienellä valmistetussa näytteessä maitohapon määrä taas lisääntyi. Tämä oli tiedettävästi ensimmäinen kerta, kun tutkittiin härkäpaputempen valmistusta Rhizopus -homesienen ja propionibakteerin yhteisfermentaatiolla B12-vitamiinin tuottamiseksi. Tutkimus antoi lupaavia tuloksia B12-vitamiinipitoisen tempen valmistuksesta, sillä härkäpaputempejen vitamiinipitoisuudet olivat huomattavasti suurempia kuin mitä aikaisemmissa tutkimuksissa soija- ja lupiinitempeillä homesienen ja propionibakteerin yhteisfermentaatiossa saadut vitamiinipitoisuudet.
  • Aalto, Sanni (2017)
    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.
  • Kankfelt, Sampo (2024)
    Vitamin B12 is crucial for human well-being, and its intake from diet primarily comes from animal-derived products. Cow's milk is a good source of B12 for humans due to its high B12 content, and it is essential to investigate whether the vitamin concentration can be influenced, for example, by the animal's diet. The aim of the thesis was to determine the nutritional and metabolic factors influencing the B12 concentration in cow's milk. The meta-analysis dataset consisted of three experiments conducted in the Viikki Research Farm from 2019 to 2022. The study subjects were primiparous and multiparous Ayrshire cows. The average days in milk was 149 days. Proportion of concentrate in the cows' diets varied from 40 to 45 %. The dataset consisted of individual observations and contained 166 different variables, with number of observations ranging from 64 to 97. Variables included dry matter intake, nutrient intakes and digestibilities, concentrations of plasma metabolites and insulin, milk yield, and composition of milk, including fatty acids. The average vitamin B12 content of milk was 3.34 ng/g (1.33–6.77 ng/g). Relationships between the studied variables were analyzed using SAS software, employing correlation and regression analysis. The results indicated that the factors with highest correlation coefficient with milk B12 concentration were the stage of lactation, plasma insulin concentration, and milk protein, fat, and dry matter concentration. The best explanatory power (R2=0.597) was achieved with a regression model where milk B12 concentration was explained by milk fat content, plasma insulin concentration, and the stage of lactation. Contrary to the hypothesis, no significant relationships were observed between dietary fiber and starch contents and milk B12 vitamin concentration. Results might have been influenced by the small size of the dataset and relatively small variation in the compositions of the diets. Due to the limited variation in diet compositions, the dataset might not have been ideally suited for the study of nutritional factors. The meta-analysis did not reveal clear nutritional strategies to increase the B12 vitamin concentration in milk. However, the study highlighted the plasma insulin concentration, which positively correlated with the milk B12 vitamin concentration. The relationship between plasma insulin and milk B12 vitamin concentration presents an interesting subject for further research.
  • SIhvola, Jenni (2022)
    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).
  • Mattila, Tuulia (2020)
    P. freudenreichii has long been used as a secondary starter culture in the production of emmental cheeses and similar Swiss-type cheeses and it is capable of producing human active form of vitamin B12. P. freudenreichii is a traditional bacterium of the dairy group, but it has also been isolated from plant products such as barley. The literature section of the dissertation dealt with propionic acid bacteria and especially P. freudenreichii as well as vitamin B12 and its analytics. The purpose of the experimental part of the dissertation was to investigate the salt and acid tolerance of P. freudenreichii isolates. In addition, the extent to which P. freudenreichii isolates grow on a plant-derived medium and ferment vitamin B12 therein was investigated. The liquid chromatographic UHPLC method validated for vitamin B12 was used to determine vitamin B12. The P. freudenreichii isolates studied in this study did not tolerate acidic conditions but grew better in a neutral or slightly alkaline environment. They also did not tolerate high NaCl concentrations and grew better in lower the salt concentrations. P. freudenreichii isolates grew well in the wheat bran matrix. The best-growing isolates were assayed for vitamin B12 production. The isolates studied produced significant amounts of vitamin B12 in plant-derived medium.
  • Anttila, Johannes (2022)
    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.
  • Hiippala, Kaisa (2014)
    The literature review dealed with the energy metabolism of Propionibacterium freudenreichii with a particular focus on carbon substrates and metabolic end-products. Also, carbon sources and other factors affecting the efficiency of vitamin B12 production by Propionibacterium were studied, including different food related matrices as a source of growth substrates. Cereal matrix as a possible growth substrate was discussed. The aim of the experimental part was to study the effect of different carbon sources on the B12 yield and metabolism of three P. freudenreichii strains of dairy or cereal origin. After preliminary screening, five carbon sources (lactate, glucose, lactose, fructose and inositol) and three strains were selected for the fermentation process, which consisted of anaerobic (72 h) and aerobic incubation (96 h) at 30 °C. The phosphate-buffered medium (0.1 M) contained only tryptone, yeast extract, CoCl2 and a carbon source (pH 6.8). Optical densities and pH values were measured at two time points and cell masses after the fermentation. Vitamin B12 produced by the strains was extracted from the fermentation samples and analysed using UHPLC method. Also consumed riboflavin was determined from the culture broths with UHPLC. Carbon consumption and metabolic end-products were analysed with HPLC after both stages of fermentation. Differences in fermentation and growth patterns between the strains were observed depending on the carbon source. Strain 1 and 2 produced the highest amounts of cell mass (396 and 589 mg, fw) fermenting fructose. Moreover, the carbon consumption was slower with over half of fructose remaining in the medium after the anaerobic phase. In addition, the pH value did not drop as quickly as in the other sugar media. When B12 yield was measured per gram cells, lactate was the most profitable carbon source for strains 1, 2 and 3 (100, 115 and 58 ?g/g cells, respectively). Yet, strains 1 and 2 synthesised the most vitamin B12 catabolising fructose when the yield was calculated per mL medium (958 and 731 ng/mL). In fructose medium both strains also produced less inhibitory metabolite, propionate. Strain 3 grew poorly during the fermentation, which affected the metabolite production. As a conclusion, fructose, besides lactate and glucose, appeared as a suitable carbon substrate for the B12 production by P. freudenreichii but the effect of fructose on its metabolism should be further investigated.
  • Hovilehto, Emmi (2014)
    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.