Browsing by Subject "UHPLC"
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(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.
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(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.
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(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.
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(2019)A common quality issue in fresh-cut vegetables is enzymatic browning. Anti-browning agents regulated by (EU) N:o 1129/2011 can be used in processing and vegetables are usually treated with compounds after washing and cutting. Polyphenol oxidase enzyme (PPO) in vegetables ox-idises phenolic compounds to quinones and brown melanin pigments are formed when reactive quinones polymerize. The literature review focused on phenolic compounds of apples, the mech-anism of action and inhibition of PPO. In addition, the review covered anti-browning agents in-vestigated in previous studies and physical inhibition methods for enzymatic browning. The aim of the experimental study was to find a mixture of anti-browning agents classified as a processing aid for fresh-cut apple with the help of the experimental design. In addition, apples treated with three different concentrations of calcium ascorbate were also investigated. The colour of the treated apples was measured using a colorimeter, the activity of PPO was determined spec-trophotometrically and the concentrations of the most common phenolic compounds of Granny Smith were determined by Ultra-High Performance Liquid Chromatography (UHPLC). The concentrations of anti-browning agents of the mixtures in PLSR model did not have a statis-tically significant effect on colour and the coefficient of determination was satisfactory. The ap-ples from the experimental design maintained their colour over one but less than five days and the apples treated with calcium ascorbate maintained their colour for one week which was the targeted shelf life. The PPO activity of the samples from the experimental design increased during the storage but activity of the samples treated with calcium ascorbate was zero all the time. The standard deviations of concentrations of phenolic compounds were high and no clear trend re-garding the change of concentrations and browning was noticed. The three major phenolic com-pounds of Granny Smith were (-)-epicatechin, (+)-catechin and chlorogenic acid. The concentra-tions of catechins were higher in the peel than in the flesh but for chlorogenic acid it was the opposite. The shelf life of one week was not achieved with the mixtures of the experimental design but calcium ascorbate acted effectively. Also compounds in the mixtures had the ability to inhibit the PPO because the apples did not start to brown immediately as the control samples treated with water. The results confirmed previous results from the literature that calcium ascorbate is an effective anti-browning agent and therefore it is one of the most common anti-browning agents in the fresh-cut industry.
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(2019)Avenanthramides are hydroxycinnamic acids unique to oat (Avena sativa L., Poaceae). They consist of an anthranilic acid part (anthranilic acid or hydroxylated and/or methoxylated derivative of anthranilic acid) that is conjugated to a cinnamic acid part via an amide bond. More than 25 different avenanthramides are found and identified in oat. However, the most common forms are esters of 5-hydroxyanthranilic acid with caffeic (2c), ferulic (2f) and p-coumaric (2p) acids. Avenanthramides have been shown to possess antioxidant and anti-inflammatory properties. Currently there is a great interest towards oat and bioactive compounds of oat, like avenanthramides. In the previous studies there has been a lot of diversity concerning the extraction method used for analysis of avenanthramides and usually quantitation methods were based on high-performance liquid chromatography (HPLC). The aim of this research was to develop a method based on ultra-high-performance liquid chromatography (UHPLC) for the analysis of the most common forms of avenanthramides (2c, 2f and 2p) in oat and oat products. In addition, also other forms of avenanthramides, like 2fd and 2pd, were identified and quantified using liquid chromatography-mass spectrometry (UHPLC-QTOF-MS). The extraction method of avenanthramides was optimized and the UHPLC-PDA method used for quantitation was validated. Finally, the study measured the differences in concentrations of avenanthramides in different oat products. In this study it was recognized that the concentrations and the extent of variation of the concentrations of avenanthramides were affected by the sample amount, the homogeneity of the samples and the extraction time used. Especially bigger sample amount of oat flour (0.5 g) led to larger and more reproducible results than smaller amount (0.1 g). The ratio of sample and solvent 1:10 worked excellently and as an extraction solvent ethanol:water (80:20) was more efficient than tested ethanol:water (80:20) with a phosphate buffer at pH 2.8. Smaller particle size of oat flour and extraction overnight led to better extractability of avenanthramides than a short extraction of sample with larger particle size. The UHPLC method was optimized using chromatographic parameters. The avenanthramides separated from each other acceptably and were eluted as follows during the 16-minute run: 2c, 2p, 2f. The response of the UHPLC instrument was linear in the tested concentration range for all three avenanthramides. The results were reproducible, and the accuracy of the UHPLC instrument was acceptable. The recovery % for avenanthramides were 99–117%. Also, other forms of avenanthramides, like 2fd, 2pd, 5p, 5f ja 3f, were identified in different oat samples using UHPLC-QTOF-MS technique. The total amount of avenanthramides in analyzed oat samples varied between 3–41.3 µg/g per fresh weight. Oat bran included them the most and oat snack the least. Avenanthramide 2c was dominant in oat flour which included 2p the least. In oat bran, in oat drink, in oat snack, in Nyhtökaura and in Muru the avenanthramide 2f was dominant over the two other forms. The UHPLC method developed in this study can be applied to the analysis of the most common forms of avenanthramides 2c, 2f and 2p in oat and in oat products and can be used in oat research in the future. The method can also be used to identify and quantitate more widely other forms of avenanthramides in different oat products. The optimized extraction was shown to be functional and reproducible to already homogeneous and processed samples, but the extraction of non-homogeneous samples could be optimized further in the future.
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(2023)Wood-decaying fungi of Basidiomycota are important decomposers of wood and plant biomass in nature. Interactions of five birch-wood decaying fungi (Fomitopsis pinicola, Fp; Fomitopsis betulina, Fb; Phlebia radiata, Pr; Fomes fomentarius, Ff; Schizophyllum commune, Sc) were studied by forming combinations of two, three or five fungal species and cultivating them on low-nitrogen liquid medium, and on three different agar media. Of the fungal species studied, Sc was the most dominant at the metabolic level. Pr was more dominant than Fp, Ff and Fb, while Fp was more dominant than Ff and Fb. This conclusion was supported by measurements from the cultivations on liquid medium (pH, enzyme activities, antioxidant and reduction ability, sugar consumption), non-metric multidimensional scaling (NMDS) analysis and compounds found in extracts from culture fluids and mycelia. Three variations of mycelial interactions were observed when fungal combinations were cultivated on agar media: inhibition and two types of co-existence. Tens of compound peaks were detected in HPLC-chromatograms of the culture fluid and mycelial extracts, but only a part of the compounds could be identified. The presence of two or more fungal species in the cultivations increased the number of peaks in the chromatograms indicating the potential of co-cultivation as a method for production of new natural compounds. Several extracts showed weak antibacterial properties. These results suggest that larger-scale cultivations, more concentrated extracts, and isolation of the numerous compounds in the extracts could facilitate finding and identifying antimicrobial substances produced by fungi.
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