Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC ESI-MS. It was discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis Novel miniaturized mass spectrometric ionization techniques based on atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were studied and evaluated in the analysis of environmental samples and biosamples. The three analytical systems investigated here were gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry (GC-µAPCI-MS) and gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry (GC-µAPPI-MS), where sample pretreatment and chromatographic separation precede ionization, and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS), where the samples are analyzed either as such or after minimal pretreatment. The gas chromatography-microchip atmospheric pressure ionization-mass spectrometry (GC-µAPI-MS) instrumentations were used in the analysis of polychlorinated biphenyls (PCBs) in negative ion mode and 2-quinolinone-derived selective androgen receptor modulators (SARMs) in positive ion mode. The analytical characteristics (i.e., limits of detection, linear ranges, and repeatabilities) of the methods were evaluated with PCB standards and SARMs in urine. All methods showed good analytical characteristics and potential for quantitative environmental analysis or bioanalysis. Desorption and ionization mechanisms in DAPPI were studied. Desorption was found to be a thermal process, with the efficiency strongly depending on thermal conductivity of the sampling surface. Probably the size and polarity of the analyte also play a role. In positive ion mode, the ionization is dependent on the ionization energy and proton affinity of the analyte and the spray solvent, while in negative ion mode the ionization mechanism is determined by the electron affinity and gas-phase acidity of the analyte and the spray solvent. DAPPI-MS was tested in the fast screening analysis of environmental, food, and forensic samples, and the results demonstrated the feasibility of DAPPI-MS for rapid screening analysis of authentic samples.

Development of analytical techniques for studies on dispersion of actinides in the environment and characterization of environmental radioactive particles

Radioactive particles from three locations were investigated for elemental composition, oxidation states of matrix elements, and origin. Instrumental techniques applied to the task were scanning electron microscopy, X-ray and gamma-ray spectrometry, secondary ion mass spectrometry, and synchrotron radiation based microanalytical techniques comprising X-ray fluorescence spectrometry, X-ray fluorescence tomography, and X-ray absorption near-edge structure spectroscopy. Uranium-containing low activity particles collected from Irish Sea sediments were characterized in terms of composition and distribution of matrix elements and the oxidation states of uranium. Indications of the origin were obtained from the intensity ratios and the presence of thorium, uranium, and plutonium. Uranium in the particles was found to exist mostly as U(IV). Studies on plutonium particles from Runit Island (Marshall Islands) soil indicated that the samples were weapon fuel fragments originating from two separate detonations: a safety test and a low-yield test. The plutonium in the particles was found to be of similar age. The distribution and oxidation states of uranium and plutonium in the matrix of weapon fuel particles from Thule (Greenland) sediments were investigated. The variations in intensity ratios observed with different techniques indicated more than one origin. Uranium in particle matrixes was mostly U(IV), but plutonium existed in some particles mainly as Pu(IV), and in others mainly as oxidized Pu(VI). The results demonstrated that the various techniques were effectively applied in the characterization of environmental radioactive particles. An on-line method was developed for separating americium from environmental samples. The procedure utilizes extraction chromatography to separate americium from light lanthanides, and cation exchange to concentrate americium before the final separation in an ion chromatography column. The separated radiochemically pure americium fraction is measured by alpha spectrometry. The method was tested with certified sediment and soil samples and found to be applicable for the analysis of environmental samples containing a wide range of Am-241 activity. Proceeding from the on-line method developed for americium, a method was also developed for separating plutonium and americium. Plutonium is reduced to Pu(III), and separated together with Am(III) throughout the procedure. Pu(III) and Am(III) are eluted from the ion chromatography column as anionic dipicolinate and oxalate complexes, respectively, and measured by alpha spectrometry.

F-18 Labelling Synthesis, Radioanalysis and Evaluation of A Dopamine Transporter and A Hypoxia Tracer

Positron emission tomography (PET) is an imaging technique in which radioactive positron-emitting tracers are used to study biochemical and physiological functions in humans and in animal experiments. The use of PET imaging has increased rapidly in recent years, as have special requirements in the fields of neurology and oncology for the development of syntheses for new, more specific and selective radiotracers. Synthesis development and automation are necessary when high amounts of radioactivity are needed for multiple PET studies. In addition, preclinical studies using experimental animal models are necessary for evaluating the suitability of new PET tracers for humans. For purification and analysing the labelled end-product, an effective radioanalytical method combined with an optimal radioactivity detection technique is of great importance. In this study, a fluorine-18 labelling synthesis method for two tracers was developed and optimized, and the usefulness of these tracers for possible prospective human studies was evaluated. N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane ([18F]β-CFT-FP) is a candidate PET tracer for the dopamine transporter (DAT), and 1H-1-(3-[18F]fluoro-2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) is a well-known hypoxia marker for hypoxic but viable cells in tumours. The methodological aim of this thesis was to evaluate the status of thin-layer chromatography (TLC) combined with proper radioactivity detection measurement systems as a radioanalytical method. Three different detection methods of radioactivity were compared: radioactivity scanning, film autoradiography, and digital photostimulated luminescence (PSL) autoradiography. The fluorine-18 labelling synthesis for [18F]β-CFT-FP was developed and carbon-11 labelled [11C]β-CFT-FP was used to study the specificity of β-CFT-FP for the DAT sites in human post-mortem brain slices. These in vitro studies showed that β-CFT-FP binds to the caudate-putamen, an area rich of DAT. The synthesis of fluorine-18 labelled [18F]FMISO was optimized, and the tracer was prepared using an automated system with good and reproducible yields. In preclinical studies, the action of the radiation sensitizer estramustine phosphate on the radiation treatment and uptake of [18F]FMISO was evaluated, with results of great importance for later human studies. The methodological part of this thesis showed that radioTLC is the method of choice when combined with an appropriate radioactivity detection technique. Digital PSL autoradiography proved to be the most appropriate when compared to the radioactivity scanning and film autoradiography methods. The very high sensitivity, good resolution, and wide dynamic range of digital PSL autoradiography are its advantages in detection of β-emitting radiolabelled substances.

Development of Sample Pretreatment and Liquid Chromatographic Techniques for Antioxidative Compounds

In this study, novel methodologies for the determination of antioxidative compounds in herbs and beverages were developed. Antioxidants are compounds that can reduce, delay or inhibit oxidative events. They are a part of the human defense system and are obtained through the diet. Antioxidants are naturally present in several types of foods, e.g. in fruits, beverages, vegetables and herbs. Antioxidants can also be added to foods during manufacturing to suppress lipid oxidation and formation of free radicals under conditions of cooking or storage and to reduce the concentration of free radicals in vivo after food ingestion. There is growing interest in natural antioxidants, and effective compounds have already been identified from antioxidant classes such as carotenoids, essential oils, flavonoids and phenolic acids. The wide variety of sample matrices and analytes presents quite a challenge for the development of analytical techniques. Growing demands have been placed on sample pretreatment. In this study, three novel extraction techniques, namely supercritical fluid extraction (SFE), pressurised hot water extraction (PHWE) and dynamic sonication-assisted extraction (DSAE) were studied. SFE was used for the extraction of lycopene from tomato skins and PHWE was used in the extraction of phenolic compounds from sage. DSAE was applied to the extraction of phenolic acids from Lamiaceae herbs. In the development of extraction methodologies, the main parameters of the extraction were studied and the recoveries were compared to those achieved by conventional extraction techniques. In addition, the stability of lycopene was also followed under different storage conditions. For the separation of the antioxidative compounds in the extracts, liquid chromatographic methods (LC) were utilised. Two novel LC techniques, namely ultra performance liquid chromatography (UPLC) and comprehensive two-dimensional liquid chromatography (LCxLC) were studied and compared with conventional high performance liquid chromatography (HPLC) for the separation of antioxidants in beverages and Lamiaceae herbs. In LCxLC, the selection of LC mode, column dimensions and flow rates were studied and optimised to obtain efficient separation of the target compounds. In addition, the separation powers of HPLC, UPLC, HPLCxHPLC and HPLCxUPLC were compared. To exploit the benefits of an integrated system, in which sample preparation and final separation are performed in a closed unit, dynamic sonication-assisted extraction was coupled on-line to a liquid chromatograph via a solid-phase trap. The increased sensitivity was utilised in the extraction of phenolic acids from Lamiaceae herbs. The results were compared to those of achieved by the LCxLC system.

Computational Techniques for Haplotype Inference and for Local Alignment Significance

This thesis which consists of an introduction and four peer-reviewed original publications studies the problems of haplotype inference (haplotyping) and local alignment significance. The problems studied here belong to the broad area of bioinformatics and computational biology. The presented solutions are computationally fast and accurate, which makes them practical in high-throughput sequence data analysis. Haplotype inference is a computational problem where the goal is to estimate haplotypes from a sample of genotypes as accurately as possible. This problem is important as the direct measurement of haplotypes is difficult, whereas the genotypes are easier to quantify. Haplotypes are the key-players when studying for example the genetic causes of diseases. In this thesis, three methods are presented for the haplotype inference problem referred to as HaploParser, HIT, and BACH. HaploParser is based on a combinatorial mosaic model and hierarchical parsing that together mimic recombinations and point-mutations in a biologically plausible way. In this mosaic model, the current population is assumed to be evolved from a small founder population. Thus, the haplotypes of the current population are recombinations of the (implicit) founder haplotypes with some point--mutations. HIT (Haplotype Inference Technique) uses a hidden Markov model for haplotypes and efficient algorithms are presented to learn this model from genotype data. The model structure of HIT is analogous to the mosaic model of HaploParser with founder haplotypes. Therefore, it can be seen as a probabilistic model of recombinations and point-mutations. BACH (Bayesian Context-based Haplotyping) utilizes a context tree weighting algorithm to efficiently sum over all variable-length Markov chains to evaluate the posterior probability of a haplotype configuration. Algorithms are presented that find haplotype configurations with high posterior probability. BACH is the most accurate method presented in this thesis and has comparable performance to the best available software for haplotype inference. Local alignment significance is a computational problem where one is interested in whether the local similarities in two sequences are due to the fact that the sequences are related or just by chance. Similarity of sequences is measured by their best local alignment score and from that, a p-value is computed. This p-value is the probability of picking two sequences from the null model that have as good or better best local alignment score. Local alignment significance is used routinely for example in homology searches. In this thesis, a general framework is sketched that allows one to compute a tight upper bound for the p-value of a local pairwise alignment score. Unlike the previous methods, the presented framework is not affeced by so-called edge-effects and can handle gaps (deletions and insertions) without troublesome sampling and curve fitting.