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Foodborne Staphylococcus Aureus: Identification and Enterotoxin Production in Milk and Cheese.
Hrušková, Vendula ; Španová, Alena (referee) ; Kmet,, Vladimír (referee) ; Kaclíková, Eva (advisor)
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.
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Elemental analysis of cheese with white mold on the surface
Pavlechová, Alžbeta ; Vítová, Eva (referee) ; Diviš, Pavel (advisor)
This Master's thesis deals with elementary analysis of cheeses with white mold on the surface. Theoretical part describes general characteristics of cheese, classification of cheeses, their nutritional aspects and characteristics of cheeses with white mold on the surface. Large part is focused on description of mineral substances selected for analysis. At the end of the theoretical part, the Kjeldahl method and emission spectrometry with inductively coupled plasma (ICP-OES) were described. Experimental part deals with preparation of samples, approach of analysis and processing of measured results. Eleven samples of cheeses were used for analysis. These samples were from Czech Republic, France and Slovak Republic. Kjeldahl method was used to determine protein contents and ICP-OES was used for elemental analysis. The results were compared to each other and also with results from other studies. Subsequently, they were statistically processed and evaluated in the final part.
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PCR identification of nonpathogenic bacteria strains in cheeses
Jurečková, Nela ; Doušková, Dagmar (referee) ; Španová, Alena (advisor)
Different species of genus Bifidobacterium are part of human and animal intestinal flora. These bacteria have benefit effects and therefore they are used in foods and pharmaceutical products as probiotics. Cheese is now suitable as a probiotic matrix except yoghurts and fermentated milks. This diploma thesis was focused on optimalization of DNA isolation from bacteria of genus Bifidobacterium. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for DNA isolation in presence of 8% polyethyleneglycol PEG 6000 and 5 M sodium chloride. Phenol extraction weas also used as an isolation method. Isolated DNA was used for amplification in domain, genus and species specific PCRs. Optimized method was tested for detection of bacteria of genus Bifidobacterium in experimentaly prepared probiotic cheeses. These cheeses contained potential probiotic bacteria from Laktoflóra collection. Bacteria were identified into species using species specific PCR. Species Bifidobacterium animalis was identified in all samples of probiotic cheeses.
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The addition of carbon dioxide to milk for cheese production
Králová, Petra ; Hrstka, Miroslav (referee) ; Kvasnička, Miloš (advisor)
This work deals with the addition of carbon dioxide to milk for cheese production. The first aim was to elaborate a literary search focused on chemical composition of milk and its changes after addition of carbon dioxide and to describe production processes, with or without addition of carbon dioxide. The opening part was in particular focused on production of cheese with carbon dioxide and what all this gas causes during production. Carbon dioxide significantly decreases pH, thereby preventing from multiplication of undesirable microorganisms, it is also very important for shortening of production time, because it shortens the coagulation time. The main aim of experimental part was to determine the optimum addition of carbon dioxide to milk for production of Balkan cheese and using several analytical methods to compare cheese produced with and without addition of carbon dioxide. The sensory methods suitable for following of sensory differences of produced cheeses were also chosen and applied. By request of Dairy in Polná Ltd., the operation economy was calculated, which revealed that production with carbon dioxide is very useful and can reduce production costs of Balkan cheese, which would certainly be positive. The advantages and disadvantages of cheese production with carbon dioxide for consumers as well as for producer are evaluated as conclusion of this work.
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DNA analysis of nonpathogenic clostridia isolated from cheeses
Chroboková, Maria ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is a molecular method which allows in vitro replication of nucleic acids. It allows the identification and quantification of microorganisms or to prove specific gene sequentions in different matrices of biological origin. Some nonpathogenic species of genus Clostridium cause damages of cheeses, so their identification and quantification is very important in cheesemaking. In this thesis, specific primers for genus Clostridium were tested. Bacterial DNA from culture collection strains and from strains isolated from damaged cheeses were used. Genus-specific region for Clostridium was amplified using specific primers. The PCR products (619 bp) were detected using electrophoresis in 1,8% agarose gel. Genus-specific character of primers was confirmed. DNA of Lactobacillus was used for negative control.
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Technology for the production of wine sausage with the addition of cheese
Kejmarová, Anna ; Nábělek, Jakub (referee) ; Mikulíková, Renata (advisor)
The aim of the bachelor thesis was to investigate the effect of soft cheese on the organoleptic properties of wine sausage. Samples of wine sausages were taken before and after baking and the percentage of lipids in the sample and the content of individual fatty acids were determined using the analytical method of gas chromatography. The sensory analysis was divided into two parts. In the first part, the evaluators assessed the appearance, aroma, consistency, texture, taste, overall evaluation and balance of the cheeses. The second part focused on the overall evaluation and selection of the most flavourful samples. The data obtained from the sensory analysis were processed and evaluated using Excel. The most unpalatable wine sausage, from the results of the sensory analysis, was the wine sausage with ermine.
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Dairy products in human nutrition, labelling according to czech legislation; consumer information and preferences
Dovolová, Barbora ; Kudlová, Eva (advisor) ; Dostálová, Jana (referee)
This bachelor thesis evaluates the dairy products labelling in the Czech Republic based on the requirements of the Czech legislation. It also pursues consumer's taste preferences, their interest and awareness of the dairy products labelling. The theoretical section mentions the history of dairying and the importance of milk products in human nutrition. The following chapters present individual dairy products and it described their production, labeling and division into kinds. The practical section reveals that the dairy products are labeled in accordance with the Czech legislation. The results are presented in the form of photos and tables (mini photobook). This part also examines whether the adult people interest in the dairy product labeling, how to orient in a range of dairy products and which products they prefer. The product evaluation was carried out in two big stores in Jablonec nad Nisou by using photographs. It was found that majority of companies indicates their milk products correctly. Identified deviations were discovered in the labelling of nutrition declaration or allergens. This is a relatively new requirements but very important. The questionnaire was found that 68 percent of people read informations on dairy products voluntarily. Most of the respondents consume especially milk, yoghurts and...
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Vliv protektivních mikrobiálních kultur na senzorickou kvalitu sýrů
Urbanová, Iveta
The thesis is aimed at lactic acid bacteria with protective effects. In the theoretical part of the thesis, the selected species of protective cultures are characterised. Next, the thesis deals with the antimicrobial and antifungal activity of protective cultures. It also provides information about specific metabolites which are produced by these cultures. Emphasis is put on the effect of protective cultures on the sensory properties of cheeses. The thesis also includes a chapter on the production of semi-hard cheeses. In the practical part of the thesis, the effect of protective cultures on the sensory quality of semi-hard cheeses is observed. The sensory analysis is performed on cheeses with protective cultures and without these cultures. Differences are observed in appearance, colour, odour, texture and taste. The sensory quality of the cheeses was monitored four months. According to the results, there were no negative effects on the sensory properties of the cheeses at the end of the monitored period.
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