Foodborne Staphylococcus Aureus: Identification and Enterotoxin Production in Milk and Cheese.

Onemocnění z potravin (alimentární onemocnění) vyvolaná bakteriemi jsou stále aktuálním tématem v celosvětovém měřítku. Abychom zajistili výrobu zdravotně nezávadných potravin, je potřeba nových poznatků o virulenci patogenů, které by doplnily již známé skutečnosti o jejich růstu a přeživání v potravinách. Také potřebujeme vyvíjet rychlé a citlivé metody na detekci těchto patogenů. Dizertační práce popisuje metodu na detekci S. aureus v potravinách, která je založená na PCR v reálném čase ve spojení s namnožením v selektivním médium. Dále pojednává o vlivu environmentálních faktorů na růst S. aureus a tvorbu enterotoxinů v mléce a sýrech. Vyvinuli jsme rychlou a citlivou metodu na detekci S. aureus v potravinách s použitím selektivního namnožení a PCR v reálném čase. Nově vyvinutá metoda umožnila detekci S. aureus na druhý den od přijetí vzorku. Tato metoda může být použita jako rychlejší, citlivějsí a vysoce specifická alternativní metoda ke konvenční mikrobiologické metodě. Zkoumali jsme vliv tří různých teplot, 8°C, 12°C a 20°C na růst S. aureus a tvorbu enterotoxinu D v pasterizovaném mléce a na růst, expresi genu sed a tvorbu enterotoxinu D v tekutém médiu s extraktem z mozku a srdce (BHI). Experimenty byly prováděny v malých skleněných fermentorech po 6 dní. Genová exprese byla sledována pomocí qRT-PCR a tvorba enterotoxinu D byla měřena pomocí imunologické metody ELISA. Růstová křivka v BHI měla stejný průběh při 20°C a 12°C, ale v při 12°C začal růst se spožděním. Při 8°C nebyl pozorován žádný růst. Růst S. aureus v mléce byl ve srovnání s BHI menší. sed mRNA byla detekována při 20°C po 4 hodinách a při 12°C po 7 hodinách a produkce enterotoxinu se objevila v exponenciální fázi růstu. V mléce se produkce SED při 20°C a při 12°C objevila dříve, ale celkové množství vyprodukovaného SED bylo nižší než v BHI. Při 8°C nebyla pozorována žádná produkce SED stejně jako v BHI. Dále byl zkoumán společný vliv nízké teploty 12°C a přítomnosti kompetitivní doprovodné mikroflóry pocházející ze surového mléka na růst S. aureus a produkci enterotoxinu v pasterizovaném mléce. Byl pozorován inhibiční účinek na růst a produkci enterotoxinů a vliv kompetice byl výraznější než vliv nízké teploty. Produkce enterotoxinu byla nízká a odpovídala růstu. Snížením množství doprovodné mikroflóry a zvýšením inokula došlo pouze k nepatrnému zvýšení produkce enterotoxinu. V další fázi byly dva různé typy sýrů zaočkovány S. aureus za účelem simulace sekundární kontaminace při výrobě sýrů. Vzorky byly odebírány v průběhu 4 týdnů. Kritické faktory jako jsou kompetitivní mikrofóra nebo pH, které jsou zodpovědné za regulaci virulence S. aureus byly sledovány. Snažili jsem se rozlišit situace při kterých: (i) není pozorován růst, ale objevuje se produkce enterotoxinu a (ii) dochází k růstu ale bez produkce enterotoxinu.Foodborne diseases caused by bacteria are an actual issue worldwide. To produce food, which is safe for human consumption, data about food-borne pathogen virulence is required to complement the already existing knowledge about the bacterial growth and survival in food. There is also a growing need for rapid and sensitive methods to detect these pathogens. In this dissertation, the real-time PCR-based method for the detection of S. aureus in food using selective enrichment and the impact of environmental factors on S. aureus growth and enterotoxin production in milk and cheese are described. We developed a rapid and sensitive method for the detection of S. aureus in food using selective enrichment and a new species-specific real-time PCR. The method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to the microbiological method. We investigated the effect of three different temperatures, 8°C, 12°C and 20°C on S. aureus growth and SED production in pasteurized milk and on growth, sed gene expression and SED production in Brain heart infusion. The experiments were performed in small-scale fermentors for six days and gene expression was followed by qRT-PCR. SED production was measured using Enzyme-Linked ImmunoSorbent Assay (ELISA). In BHI the growth pattern was the same at 20°C and 12°C but delayed in the latter. At 8°C there was no growth. In milk, growth was lower compared to BHI. sed mRNA was detected at 20°C and 12°C after 4 and 7 hours respectively in BHI and the production occurred during the exponential phase of growth. In milk the SED production at 20°C and 12°C occurred earlier in growth but a lower total amount was produced compared to BHI. At 8°C, there was no SED production like in BHI. The combined effect of low temperature, 12°C, and the presence of competing background microflora derived from raw milk on the growth of S. aureus and SED production in pasteurized milk was further investigated. An inhibitory effect on S. aureus growth and enterotoxin production was observed and the impact of competition was greater than the impact of low temperature. The enterotoxin production was low and correlated with the growth. By lowering the amount of competing microflora and increasing the inoculation level of S. aureus, only a slight increase in enterotoxin production occurred. In the next stage, two different cheese matrices were inoculated with S. aureus to simulate a post-contamination scenario in cheese manufacture. Samples were collected over period of 4 weeks. Critical food factors, like competing microflora and pH, which are responsible for down- and up-regulation of the virulence of S. aureus, were monitored. We tried to indentify if there are situations in which: (i) no growth but enterotoxin formation is observed, and (ii) growth and no enterotoxin formation occurs.

Continued Fractions of Quadratic Numbers

In this paper, we will first summarize known results concerning continued fractions. Then we will limit our consideration to continued fractions of quadratic numbers. The second author described periods and sometimes precise form of continued fractions of $\sqrt{N}$, where $N$ is a natural number. In cases where we were able to find such results in literature, we recall the original authors, however many results seem to be new.Comment: 13 page

Meteorological factors influencing spread of contaminated air parcels

Bakalářská práce se zabývá různými meteorologickými faktory ovlivňující šíření kontaminovaného ovzduší. Ukazuje na hlavní zdroje kontaminace ovzduší v České republice. V práci jsou vysvětleny vlivy meteorologických podmínek na stabilitu a šíření kontaminované látky v ovzduší. Bakalářská práce se také zabývá vlivem kontaminovaného ovzduší na životní prostředí a systémem ochrany před šířením nebezpečných látek v ovzduší.Bachelor thesis deals with the meteorological factors influencing spread of contaminated atmosphere. This thesis shows the main sources of the contamination of atmosphere in the Czech Republic. There are explained the factors of meteorological condition of stability and of spread contaminated materials in the atmosphere. It is engaged in factors of contaminated atmosphere to the environment and system of protection against spread of hazardous substances in the atmosphere.

Analysis of fixing nodes used in generalized inverse computation

In various fields of numerical mathematics, there arises the need to compute a generalized inverse of a symmetric positive semidefinite matrix, for example in the solution of contact problems. Systems with semidefinite matrices can be solved by standard direct methods for the solution of systems with positive definite matrices adapted to the solution of systems with only positive semidefinite matrix. One of the possibilities is a modification of Cholesky decomposition using so called fixing nodes, which is presented in this paper with particular emphasise on proper definition of fixing nodes. The generalised inverse algorithm consisting in Cholesky decomposition with usage of fixing nodes is adopted from paper [1]. In [1], authors choose the fixing nodes using Perron vector of an adjacency matrix of the graph which is only a sub-optimal choice. Their choice is discussed in this paper together with other possible candidates on fixing node. Several numerical experiments including all candidates have been done. Based on these results, it turns out that using eigenvectors of Laplacian matrix provides better choice of fixing node than using Perron vector

SCIENTISM AND EFFECTIVENESS IN TEACHING OF FOREIGN LANGUAGES: CONTENT AND LANGUAGE INTEGRATED LEARNING (CLIL)

The concept of language education process cannot be unilaterally practical nor impractically theoretical. The principle that practice is the criterion of truth of a theory is true also in the philosophy of the teaching process. This theoretically focused contribution gives a structured view on selected factors of teaching effectiveness, emphasizing pedagogical-psychological factors. The principle of scientism of the education system is analyzed from the point of view of psycho-didactics and language teaching methodology. Language teaching must fully accept the principle emphasized by the dynamics of the educational process by continuously updating knowledge, discovering contradictions and follow the principle of unity of theory and practice. In order for motivation to be a really effective factor of effectiveness, it is necessary to focus it on psycho-didactically relevant goals which are ideally set by the teacher together with their students The authors focus their attention on goal motivational orientation of language teaching which encourages and enables autonomous and self-directed student learning

Wheat flour, bread and biscuits enriched by linseed fibre - comparison of harvest year, linseed variety and addition level

Wheat flour was fortified by 2.5, 5.0 or 10 wt. per cent of linseed fibre, gained from seeds of golden flax varieties Amon and Raciol and brown one Recital (granulation 500 - 700 m), prepared from 2015 and 2016 harvests. Using analytical tests, namely sedimentation according to Zeleny and Falling Number, basic technological quality of flour composites was mostly independent on all three observed factors (harvest year, linseed variety and addition). Rheological tests included the farinograph, the extensigraph and the amylograph proofs. Enhancement by brown and yellow flax fibre significantly contributed to rise of farinograph water absorption and to dough stability shortening, directed mainly by addition level. Extensigraph curves course depended on dough resting time, higher differences between wheat control and flour composites were observed after 60 min dough resting. Linseed fibre weakened dough extensibility, and energy as area under curve also partially decreased about 3, 8 and 25% in average as portion of alternative materials in dough has risen. Compared to control, suspension viscosities of tested flour composite generally increased; the strongest effect was recorded for composite samples from harvest year 2016. During dough leavening, tested samples were differentiated according to maturograph dough resistance, and interaction of all three factors was identified. Regardless to variantion in dough machinability, specific volumes of composite bread samples were similar through whole sample set - any unequivocal trend was found. Somewhat worse vaulting of bread was calculated for buns manufactured from raw materials of 2016 harvest. Reversely, linseed fibre produced in 2015 improved crumb softness, especially at 5% enhancement (about ca 50% in average). The lowest addition of linseed fibre resctricted biscuits spread during baking in the highest extent, but rising level of enhancement suppressed elevated dough elasticity. Both cereal products were considered as acceptable for common consumers. Multivariate PCA method verified changes mainly in protein visco-elastic properties, which were reflected in bread and biscuits quality in an opposite manner. Based on this statistics, quality of wheat controls was comparable in both harvest year if related to changes induced by linssed fibre. In opposite to this, technological and consumer's parameters of flour composites and manufactured cereal products were statistically dependent of harvest year of linseed. As presumed, the lowest addition level brough the smallest changes; multiplied fortification caused gradual variation in results of all conducted proofs. Owing to high dietary fibre content in linseed fibre (over 50%), the medium dosage of the alternative material (i.e. 5% addition) could be recommended for praxis

Linseed fibre - effect on composite flour properties and cereal products quality

Wheat flour was fortified by 2.5 or 5.0 wt. per cent of linseed fibre, gained from seeds of golden flax varieties Amon and Raciol and brown one Recital (granulation 500 - 700 m), prepared from 2015 harvest. Technological quality of six flour composites was described analytically by Falling Number and Zeleny sedimentation test. Both screening methods shown a little impact on amylases activity and protein quality, respectively. Rheological tests included the farinograph, the extensigraph and the Rapid Visco Analyser (RVA) proofs. Additions of brown and yellow flax fibre significantly increased farinograph water absorption and shortened dough stability, somewhat stronger by addition of brown linseed fibre. Extensigraph curves course depended on dough resting time, higher differences between wheat control and flour composites were observed after 60 min dough resting. Linseed fibre supported dough extensibility, and energy as area under curve significantly decreased about 7 - 18%, mainly due to increasing alternative material portion in dough. In general, fibre is characterised as hydrophilic material, and pasting profiles of flour composites confirmed this experience. During dough leavening, tested samples were differentiated according to maturograph dough resistance; optimal leavening time of wheat-linseed fibre dough was shorter than control. Regardless described modifications in dough machinability, specific volumes of bread buns were similar though whole sample set. A weak worsening of buns vaulting reflected a partial dilution of dough gluten skeleton. Cut-off biscuits were characterised by gradually lowering spread ratio, correspondingly to elevated dough elasticity. Laboratory prepared elbow-pasta have the same cooking time as the control (8.0 min), and data variation could not be attributed to linseed fibre or addition level. All three cereal products were found to have acceptable sensory profiles. PCA method verified partial lowering of protein quality and pointed at tested sample differentiation according to linseed addition level

Properties and nutritional value of wheat bread enriched by hemp products

Hemp (Cannabis sativa) is annual plant that is native to China and remained as important material for food, industrial and medical purposes. As source of cannabinoids belongs to controversial, but due to its excellent nutritional profile, non-gluten protein, fat and fibre it has potential in bakery products. Addition of 5% - 20% of hemp press cake fine flour and fine wholemeal significantly increased dietary fibre content, but their influence on volume of laboratory baked bread was different. Reflecting actual dosage, both types of hemp press cake flour diminished bun sizes about 6% - 33%; volumes of bread containing hulled wholemeal were comparable to standard (mean 310 mL/100 g vs. 333 ml/100 g, respectively. Only dehulled wholemeal hemp form increased the bread specific volume (6% - 30%), especially as 10% fortification (434 mL/100 g). Six Canadian hemp products were added as 10% and 20% on wheat flour base, comprising fine hemp flour and coarse hemp powder, dehulled whole seeds, hulled hemp seeds with sea salt as well as 50% and 43% hemp protein concentrates (KP1-KP6, respectively). The higher level of KP1, KP2, KP5 and KP6, the lower bread specific volumes were determined (decrease about 9% - 48%). Soft increase in buns size caused by 10% and 20% KP3 (323 and 319 ml/100 g vs. 296 mL/100 g) was insignificant. The effect of KP4 was reversely verifiable, magnifying the parameter about 25% and 17%, respectively. In terms of protein content in bread, a level 11.75% in wheat bread has risen to approx. 14.5% and 18.0% when 10% and 20% of KP3 and KP5, respectively, was included into bread recipe. All six Canadian hemp products increased dietary fibre content in bread, mainly owing to KP4 and both protein concentrates (up to 4 and 3 times, respectively). Incorporation of hemp flour up to the level of 10% positively affected bread sensorial properties

Continued Fractions of Square Roots of Natural Numbers

In this paper, we will first summarize known results concerning continued fractions. Then we will limit our consideration to continued fractions of quadratic numbers. The second author describes periods and sometimes the precise form of continued fractions of ?N, where N is a natural number. In cases where we have been able to find such results in the literature, we recall the original authors, however many results seem to be new

Evaluation of Model Wheat/Hemp Composites

Model cereal blends were prepared from commercial wheat fine flour and 5 samples of hemp flour (HF), including fine (2 of conventional form, 1 of organic form) and wholemeal type (2 of conventional form). Wheat flour was substituted in 4 levels (5, 10, 15, 20%). HF addition has increased protein content independently on tested hemp flour form or type. Partial model cereal blends could be distinguished according to protein quality (Zeleny test values), especially between fine and wholemeal HF type. Both flour types affected also amylolytic activity, for which a relationship between hemp addition and determined level of Falling Number was confirmed for all five model cereal blends. Solvent retention capacity profiles (SRC) of partial models were influenced by both HF form and type, as well as by its addition level. Between both mentioned groups of quality features, significant correlation were proved - relationships among protein content/quality and lactic acid SRC were verifiable on p <0.01 (-0.58, 0.91, respectively). By performed ANOVA, a possibility to distinguish the HF form used in model cereal blend according to the lactic acid SRC and the water SRC was demonstrated. Comparing partial cereal models containing fine and wholemeal hemp type, HF addition level demonstrated its impact on the sodium carbonate SRC and the water acid SRC