Browsing by discipline "Elintarvikekemia"

The aim of the present study was to implement a tandem mass spectrometry (MS/MS) method for determining glycosidic bond linkage positions of neutral isomeric glucooligosaccharides. The methods for structural analysis and the fragmentation mechanisms of oligosaccharides in MS/MS-analysis was reviewed. The MS/MS-spectra of oligosaccharide contains glycosidic bond and cross ring cleavage fragment ions. The linkage position can be determined by cross ring cleavage fragment ions of cationized or anionized oligosaccharides. The study started by analyzing the typical MS/MS-spectra of model isomeric disaccharides with different linkage positions. The data was then used to assess the MS/MS-spectra of model tri- and tetrasaccharides. The similarity of MS/MS fragmentation patterns of the model disaccharides and the tri- and tetrasaccharides would enable the linkage position analysis by the applied MS/MS method. The MS/MS-analyses were carried out by using electron spray ionization with ion trap mass spectrometer in both positive and negative mode. In positive mode the oligosaccharides were analyzed as sodium and lithium adduct ions while chloride adduct ions were used in negative mode analysis. The different linkage positions of disaccharides were characterized by the different MS/MS fragmentation profiles in positive and negative ionization methods. The reducing end linkage positions of tri- and tetrasaccharides were easily identified by comparing their MS/MS fragmentation patterns with the MS/MS-spectra of model disaccharides. The other linkages in the tri- and tetrasaccharide were identified by using negative mode. In positive mode the identification of other linkages was not possible because the cross ring cleavage fragment ions were formed mostly from the reducing end. The linkages of tri- and tetrasaccharides could not be identified by MS3-analysis due to formation of isomeric glycosidic bond fragment ions as the charge can be retained in the reducing or non-reducing end. The results indicated that the applied MS/MS method was suitable for determing the glycosidic bond linkages of oligosaccharides in negative mode. In positive mode only the reducing end linkage can be determined.

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.

Tiivistelmä/Referat – Abstract The literature part was focusing on the metabolism, use, safety and analytical methods for glyphosate (2-phosphonomethylamino) acetic acid. The focus was on methods that are used to analyze glyphosate in foods. Glyphosate is the active compound of an herbicide. The experimental part was done in the Customs laboratory. The main object was to develop a method to analyze glyphosate, aminomethylphosphonic acid (AMPA) and glufosinate in food without derivatization using UPLC-MS/MS technique. The EURL-SRM QuPPe method was used as a sample treatment for durum wheat, iceberg lettuce, orange and avocado. The separation was done using high performance liquid chromatography and detected in triple quadrupole mass spectrometer using electro-spray ionization (ESI-) and multiple reaction monitoring (MRM). Four analytical columns were evaluated: Acquity® UPLC BEH HILIC, HypercarbTM Porous Graphitic Carbon, HypercarbTM Porous Graphitic Carbon and ShodexTM HILICPak VT-50 2D connected to ShodexTM HILICPak VT-50G 2A precolumn. ShodexTM HILICPak column was chosen due to its superior retention of the compounds. The mobile phase contained water, 1 % formic acid in water and acetonitrile (70:20:10). The linearity of the method was 0.01–2.2 ng/ml for glyphosate and AMPA and 0.002–2.2 ng/ml for glufosinate. The high amount of matricecompounds that coeluted with AMPA made identifying and integrating the analyte difficult to do. The retention times of glyphosate and glufosinate were shifting more than the validationcriteria ± 0.1 minutes. Isotope labeled glyphosate (1,2-13C2 15-N-glyphosate) that was added to the samples had a similar shifting in retention times which made it possible to efficiently evaluate the validation of glyphosate. The lack of internal standard for glufosinate meant that the method could not be validated for glufosinate. The limit of quantification (LOQ) for glyphosate was 0.04 µg/ml with RSD < 20. The validated method was suitable for analysing glyphosate residues in food.

The literature review of this master’s thesis dealt with polysaccharide based hydro- and aerogels and their preparation methods and characterization. TEMPO/laccase-catalyzed oxidation was also included in the literature review. The aim of the experimental part of this thesis was to prepare hydrogels by TEMPO/laccase-catalyzed oxidation and study how the oxidation changed the rheological and chemical characteristics of the polysaccharides. The hydrogels were also dried to produce hydrogels via lyophilization and their characteristics were determined. The degree of oxidation of the hydrogels was analyzed by the GC-MS method. The viscoelastic properties of the hydrogels were studied by oscillation measurements. The compressive modulus of the aerogels was determined by means of a compression test. The morphology of the aerogels was studied with the help of a scanning electron microscopy (SEM). The studied polysaccharides were arabinoxylan, glucomannan, galactomannan and xyloglucan. Due to the oxidation, a change in the viscoelastic behaviour could be seen between the native and oxidised polysaccharides. The rheological test revealed that the native polysaccharides were viscous liquids (G' > G'') and oxidized polysaccharides formed elastic hydrogels (G' > G''). The degree of oxidation of the hydrogels varied 3,3–11,7 %. The hydrogels were dried using two different freezing methods, unidirectional and conventional, and they were freeze-dried into aerogels without significant shrinkage. The density of the aerogels varied 0,017–0,030 g/mm3. The compressive modulus of the aerogels was 108‒1184 kPa depending on the polysaccharide. The SEM images revealed that unidirectional freezing resulted in pores that were parallelly oriented with the freezing direction. Unidirectionally frozen aerogels were stronger than conventionally frozen aerogels when the compression was oriented against the freezing direction. This was the first time that TEMPO/laccase catalyzed oxidation was used to prepare arabinoxylan and glucomannan hydro- and aerogels. The arabinoxylan and glucomannan aerogels were mechanically stronger than previously studied aerogels that have been prepared by enzymatic oxidation.

Diacetyl and acetoin are typical butter flavours, which are also present in other fermented dairy products such as sour milk. Diacetyl and acetoin are mainly produced from their precursor ?-acetolactate (ALA) by lactic acid bacteria as a product of their metabolism. In the literature review the formation of diacetyl and acetoin were examined along with the typical techniques to analyse diacetyl. The aim of the experimental work was to develop and validate a method for the determination of diacetyl and acetoin in sour milk by gas-chromatographic technique coupled with flame ionisation detector (GC-FID). In this study a suitable column and gas-chromatographic parameters to analyse diacetyl and acetoin were obtained and two sample preparation techniques were tested. In addition, the reactivity of diacetyl and acetoin and their interactions with the sample matrix were examined. Finally the method was validated. In the complete method, the proteins of the sample were precipitated with acetone and the sample was centrifuged. ALA along with protein-bound diacetyl and acetoin were extracted from the supernatant by solid-phase extraction (SPE), followed by the GC-FID-analysis of free diacetyl and acetoin in the SPE-permeate. GC-FID-analysis was performed using a polar ZB-FFAP-column, split-ratio 30:1 and inlet and detector temperatures 200 ?C and 250 ?C, respectively. Diacetyl and acetoin are reactive and easily evaporated, which complicated their analysis. ALA was mainly decomposed to acetoin in the hot injector. This finding resulted in extraction of ALA from the sample by SPE. In the sour milk matrix, diacetyl and acetoin were believed to appear in three different forms: free and strongly or weakly bound to proteins. The compounds were easily transformed from one form to another. Possibly, also cyclopentanone, the internal standard, was partly bound to the sample matrix. The reactivity of the compounds prevented the determination of total diacetyl and acetoin. Therefore, the method was suitable only for the determination of free diacetyl and acetoin, which, in fact, constitute the buttery aroma in sour milk. Sour milk contained more free acetoin than diacetyl. The method was found to be selective and sensitive for the determination of diacetyl and acetoin. However, the reactivity of the compounds impaired the repeatability of the results. Further research is needed to discover factors affecting the binding of diacetyl and acetoin to the sample matrix. Furthermore, different internal standards and solvents ought to be tested in place of cyclopentanone and acetoin.

Mikrolevät ovat lähinnä vesistöissä eläviä yksisoluisia tai yksinkertaisia monisoluisia kasveja, syanobakteereja tai alkueliöitä. Kyseessä on uusiutuva luonnonvara, jota parhaillaan hyödynnetään muun muassa elintarvikkeina, rehuna ja arvokkaiden biomolekyylien sekä monenlaisten lipidien tuottamisessa. Ylikriittinen hiilidioksidiuutto on potentiaalinen lipidien eristysmenetelmä, jossa ei tarvita ihmisille ja ympäristölle myrkyllisiä liuottimia. Uuton tehoa ja selektiivisyyttä voidaan optimoida vaihtelemalla uuttolämpötilaa ja painetta sekä uuton kestoa ja hiilidioksidin virtausnopeutta. Tutkielman kirjallisuuskatsauksen tavoitteena oli tarkastella teoreettisesti lipidien uuttomenetelmiä, erityisesti ylikriittistä uuttoa, sekä käydä läpi kirjallisuudessa jo esiintyvää aineistoa mikrolevien lipidien laadusta, määrästä ja näihin vaikuttavista tekijöistä. Erityistä huomiota oli tarkoitus kiinnittää kirjallisuuteen koskien kokeellisessa osassa käytettäviä levälajeja. Lisäksi tavoitteena oli tarkastella mikrolevien lipidien mahdollisia käyttösovelluksia. Tutkimuksen kokeellisen osan tavoitteena oli selvittää ylikriittisen hiilidioksidiuuton (SFE) käyttökelpoisuutta mikrolevien lipidien uuttamisessa verrattuna aiemmin käytössä olleeseen ja mikroleviä varten optimoituun kiihdytettyyn liuotinuuttoon (ASE). Tarkoituksena oli myös selvittää ylikriittisen hiilidioksidiuuton uuttoparametrien ja näytekoon vaikutusta uuton tehokkuuteen ja selektiivisyyteen sekä tutkia mikrolevien lipidikoostumusta. Kaikkien tutkittujen lipidikomponenttien SFE-uutolla saavutettu suhteellinen saanto oli poikkeuksetta pienempi verrattuna ASE-uuton saantoon. Kuitenkin vaihtelua oli runsaasti käytettäessä erilaisia uutto-olosuhteita ja etenkin levälajien välillä. Suurin osa lipidisaannosta uuttui SFE:llä jo ensimmäisen 10 minuutin aikana, jonka jälkeen uuttuminen oli vähäistä. Eikosapentaeenihapon (EPA) saanto SFE-uutolla oli suhteellisesti jonkin verran pienempää ASE-uuttoon verrattuna, mikä viittaa siihen, että EPA on kiinnittyneenä lähinnä poolisiin lipideihin, kuten fosfolipideihin tai glykolipideihin. Tutkittavien mikrolevien lipidipitoisuudet olivat melko pieniä moniin kirjallisuudessa esitettyihin lukuihin verrattuna, joten poolisten lipidien osuus kokonaislipideistä on luultavasti kohtalaisen suuri. Toisaalta vertailussa tulee huomioida se, että monissa kirjallisuudessa esitetyissä tuloksissa levät on kasvatettu lipidien kertymistä suosivissa olosuhteissa ja/tai lipidipitoisuus on määritetty gravimetrisesti eikä rasvahappojen summana. SFE-uutto vaikuttaa tehoavan paremmin neutraalilipideihin ja sellaisiin mikroleviin, joiden soluseinä ei ole erityisen vahva.