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Analysis of mycotoxins in barley and malt
Čuta, Robert ; Obruča, Stanislav (referee) ; Běláková, Sylvie (advisor)
This bachelor´s thesis deals with the mycotoxines present in cereals, especially in barley and malt. Teoretical part summarizes the knowledge of mycotoxines and their occurrence. The possibilities of mycotoxines determination are held forth in this thesis. In the experimental part an ultrasonic extraction metod of deoxynivalenol was optimized. For the deoxynivalenol determination was used a method of high-performance liquid chromatography with a mass detector (HPLC – MS/MS). This method was used for deoxynivalenol determination in barley and malt produced from this barley. The bachelor´s thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.
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Monitoring of the content of selected trichothecene mycotoxins in malting barley
Hrdinová, Lucie ; Kočí, Radka (referee) ; Běláková, Sylvie (advisor)
This master thesis deals with a monitoring of a content of the trichothecene mycotoxins deoxynivalenol, nivalenol, T-2 toxin and HT-2 toxin in malting barley using the LC-MS/MS method. The theoretical part describes general characteristics of mycotoxins and their significant producer filamentous fungus of Fusarium species. Further, important trichothecene mycotoxins and mycotoxins generally which are commonly found in malting barley were also characterized. In the theoretical part of the thesis possibilities for a determination of the mycotoxins by the chromatographic methods were presented too; the immunochemical methods were also mentioned. In the experimental section an analysis of the B type trichothecenes was optimized by LC/APCI-MS/MS and of the A type trichothecenes by LC/ESI-MS/MS. When analyzing 57 samples of different barley varieties the deoxynivalenol reached the highest values (up to 945,2 µg.kg-1), namely in the case of the Sebastian variety with corn as the fore-crop. The highest values of nivalenol, T-2 toxin and HT-2 toxin (138,4 µg.kg-1; 21,8 µg.kg-1 and 68,7 µg.kg-1 respectively) were found in the Prestige variety of barley with winter wheat as the fore-crop. Subsequently a second set of four experimental samples of the Sebastian variety of barley and malt produced from the variety with corn as the fore-crop were analysed. In this group three samples were artificially infected with the filamentous fungi of Fusarium species; the fourth sample was not artificially infected and served as a control sample. Even in the case of the artificially infected samples the deoxynivalenol reached the highest values. The master thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.
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Monitoring of the occurrence of mycotoxins in beers from market retail
Wawroszová, Simona ; Pospíchalová, Markéta (referee) ; Běláková, Sylvie (advisor)
This master thesis deals with monitoring of a content of deoxynivalenol, its metabolite deoxynivalenol-3-b-D-glucopyranoside and ochratoxin A in beer samples collected from retail market in the Czech Republic, Poland and Slovakia. The theoretical part describes general characteristics of mycotoxins, its transfer from field barely through malt to beer and its occurrence in beers. Malting process and brewing technology were also mentioned. Subsequently possibilities for a determination of the mycotoxins by the chromatografic and immunochemical method were presented. The experimental section describes analysis of 30 samples of beer. The analyses were conducted using ultra high-performance liquid chromatography with fluorimetric detection (UPLC/FLR) for ochratoxin A and high-performance liquid chromatography coupled with mass spectrometer (HPLC/MS) for deoxynivalenol and its metabolite. Ochratoxin A was detected in 25 of the 30 samples in concentration range of 0,6 - 82,5 ng·l-1. Deoxynivalenol was found in 24 of the 30 samples with concentration range of 2,29 - 12,57 ug·l-1 and deoxynivalenol-3-b-D-glucopyranoside was occure in 19 of the 30 samples in concentration range of 2,45 - 12,47 ug·l-1. It was also assessed the relationship between beer gushing and presence of mycotoxins in beer. No connection between the parameters has been found. Consequently it is not possible to predict beer gushing from the presence of mycotoxins.
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Aflatoxins in food and their influence on DNA and cell lines
Šislerová, Lucie ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
Aflatoxins present a great danger due to their high toxicity and carcinogenicity, which is not easily avoided in everyday life. Intoxication with aflatoxins causes a wide range of diseases ranging from mild diseases to organs necrosis or death. Aflatoxins mostly affect the liver, where it degrades and the formation of subsequent metabolites, which are the most toxic to the body. For this reason, their precise determination and understanding of the principle of their effect is very important. In this work, methods for monitoring and closer determination of aflatoxin effects on human cells were calibrated. The methods that were used are: MTT viability assays, fluorescence microscopy and flow cytometry. Next, the amount of aflatoxins present in different foods with different storage conditions was measured. For this analysis were used ELISA assays RIDASCREEN Aflatoxin Total and RIDA Aflatoxin column. Calibrated methods were compared with the methods already used to determine the effect of aflatoxins and the results of the ELISA tests were compared with the limits of aflatoxin levels permitted by the Czech legislation. None of the controlled foods contained above-the-limit concentration of aflatoxins, which in the Czech Republic is set at 4-10 µg/l (varies for different types of food). Foods that were poorly stored but not visibly affected by fungi showed the highest levels of aflatoxins. The LD50 value for aflatoxin B1 was determined to 12,25 µM. The type of cell death caused by aflatoxins was determined by flow cytometry and these data were further confirmed by fluorescence microscopy images.
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Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
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Moulds and residential buildings
Dvořák, Tomáš ; Trachtová, Štěpánka (referee) ; Omelková, Jiřina (advisor)
Bachelor’s thesis is aimed at moulds and residential buildings. At moulds occurrence and at conditions for their growth and reproduction. Thesis defined their distribution, morphology, metabolism and type of reproduction. Describe most important genera of moulds, their identification and procedure during their elimination. Practical part is aimed at two methods of determination in two different types of residential building, in prefab house and in single family house. The object of practical part was to determinate the genus of occurring moulds
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The determination of mycotoxins in commercial beers
Martiník, Jan ; Benešová,, Karolína (referee) ; Mgr. Marek Pernica, Ph.D (advisor)
Mycotoxins are secondary metabolites of microscopic fibrous micromycetes that are able to infect cereals. The use of contaminated materials can lead to transfer of mycotoxins into the final product, such as beer. The master’s thesis deals with the determination of mycotoxins in beers. The theoretical part of this thesis describes selected mycotoxins, their occurrence, toxic properties and legislative limits of the European Union. The theoretical part also deals with the description of beer production and the possibilities of mycotoxin determination. The theoretical part also describes the statistical methods used for data processing. The experimental part of this work describes the validation of the method for determination of mycotoxins in beers. This section also describes the optimization of mycotoxin extraction using a commercially available 11+Myco MS-PREP® immunoaffinity column. The conditions for the determination of mycotoxins on UPLC-MS/MS are given in this thesis. The validation parameters such as linearity, accuracy, precision, LOD and LOQ were determined. This section contains a description of beer samples used for the determination of mycotoxins. The goal of the thesis was to optimize and validate the method for determination of mycotoxins in beers. From the validation parameters, it was found that this method is suitable for its intended purpose, namely for mycotoxins aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, ochratoxin B, zearalenone, -zearalenol, -zearalenol, -zearalanol, -zearalanol, deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin and HT-2 toxin. The recoveries of this method ranged from 72,2 % to 100,0 %. The validated method was used for determination of above-mentioned mycotoxins in 89 beers. Of the total number of beers, 37 were produced in the Czech Republic and 52 in other European countries. Mycotoxins deoxynivalenol, T-2 toxin and HT-2 toxin were found in all beer samples. Common mycotoxins included fumonisin B1, ochratoxin A, -zearalenol and 3-acetyldeoxynivalenol. The mycotoxins aflatoxin B1, aflatoxin B2, aflatoxin G1, fumonisin B2, fumonisin B3, zearalenone and ochratoxin B were identified in less than 50 % of the samples. Mycotoxins aflatoxin G2, -zearalenol, -zearalanol and -zearalanol were not determined in any tested samples. The results of the analysis were subjected to statistical processing where the concentrations determined in Czech and European beers were compared. Principal component analysis and correlation analysis were created for aflatoxin B1, fumonisins, ochratoxin A, -zearalenol, deoxynivalenol, 3-acetlydeoxynivalenol, T-2 toxin and HT-2 toxin. The results of the analysis were compared with published studies.
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Mycotoxins in fermented beverages
Martiník, Jan ; Svoboda, Zdeněk (referee) ; Běláková,, Sylvie (advisor)
Mycotoxins are toxic secondary metabolites of mold and fungi that attack cereals, wine grapes or apples, which are then used to produce fermented beverages. This thesis focuses on mycotoxin patulin which is primarily found in ciders. The theoretical part describes selected mycotoxins, legislation concerning these mycotoxins and their occurrence in fermented beverages. Section of the theoretical part is also dedicated to the description of fermented beverages and analytical methods used to determine mycotoxins in beverages. The experimental part deals with the validation parameters of the method for determination of patulin and 5-HMF. It also deals with the extraction and determination of these substances. Columns EASIMIPTM PATULIN were used in the extraction of patulin and 5-HMF and the concentration was measured by ultra high performance chromatography with photodiode array detection (UPLC/PDA). The determination of patulin and 5-HMF was performed in a total of 33 samples of cider, 9 samples of apple juice, 2 samples of wine and 2 samples of radler. Patulin was found in 6,1 % of cider samples and in 44,4 % of apple juice samples. 5-HMF was found in both wines and radlers, in 78,8 % of ciders and in 77,8 % of apple juices. All measurement data is processed in the results and discussion. The results were compared with foreign studies.
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Mycotoxins in Brewing Materials and Beer
Běláková, Sylvie ; Vávrová, Milada (referee) ; Márová, Ivana (referee) ; Kráčmar, Stanislav (referee) ; Čáslavský, Josef (advisor)
The presented thesis deals with the issue of mycotoxins in brewing materials and beer. Attention was devoted mainly to the selected fusarium mycotoxins (deoxynivalenol, zearalenol, T-2 toxin, and HT-2 toxin) ochratoxin A and aflatoxins B1, B2, G1, and G2. The aim of the thesis was to optimize and validate analytical methods for the determination of the above mentioned mycotoxins in the brewing materials and beer. Analytes were separated using high-performance liquid chromatography with mass – spectrometric detection (HPLC-MS/MS) and ultra-performance liquid chromatography with fluorescence detection (UPLC/FLR). These analytical methods were then applied for mapping the occurrence of fusarium mycotoxins in malting barley crops in the Czech Republic and monitoring the level of contamination with mycotoxins in malting and brewing industries. In addition, experiments studying over-foaming of beer were conducted as primary gushing – over-foaming of beer – is connected, similarly as mycotoxins, with the presence of microscopic filamentous fungi in the raw materials for beer production. Studies describing in detail these methods are part of this thesis (Annex I – V). From all published results, it is evident that the occurrence of mycotoxins in cereals including barley is natural and cannot be completely prevented, not even if all conditions of correct agricultural practice are observed. It is known that some mycotoxins present in contaminated malting barley pass to the final product – beer due to their chemical and physical properties. However, the mycotoxin concentrations found do not mean any significant health risk for consumers.
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Stability of selected mycotoxins in beer
Štáblová, Taťána ; Benešová, Karolína (referee) ; Běláková, Sylvie (advisor)
Mycotoxins are secondary metabolites of moulds, which attack cereals, for example barley, from which mycotoxins then get to beer. This submitted work is focused on ochratoxin A, deoxynivalenol and zearalenone, which can occur in beer. The first part of this master’s thesis consists of literary research, which describes mycotoxins in general, points out their occurrence, prevention of their formation and delivers information about their physical and chemical properties and toxicity. Furthermore, the research contains basis of malt and beer technology, the occurrence of mycotoxins in beer and raw materials for its production. The research describes changes in concentration of mycotoxins across malt and beer production. The next part deals with possibilities of determination of mycotoxins in barley, malt and beer, compares individual methods of their determination and points out many difficulties of some analyses. The experimental part of this work pursues determination of ochratoxin A, deoxynivalenol and zearalenone in different types of beer with the help of UPLC-FLR, HPLC-MS and ELISA. Instrumental techniques are validated and gathered results are compared with the results in literature. The goal of this master’s thesis is to assess the stability of ochratoxin A and deoxynivalenol in beer over time. The gained results show that there are changes in the concentration of ochratoxin A over time, nevertheless those changes show no pattern. Overall, there was a decrease in concentration in 47 % of the samples and an increase in 28 % of them. In the rest of the samples the concentration did not change. The concentration of deoxynivalenol does not change over time. One of the other goals of this thesis is monitoring of selected mycotoxins in beer. The average concentration of ochratoxin A in the samples was 39 ng/l and deoxynivalenol 9,9 g/l. Zearalenone did not occur in any of the samples when determined by liquid chromatography. All results agree with literature. Next, the thesis compares different analytical methods for determination of ochratoxin A, deoxynivalenol and zearalenone. The screening method ELISA is compared to UPLC-FLR and HPLC-MS. The determination of ochratoxin A by ELISA has shown to be time consuming, nevertheless the results responded to instrumental technique. ELISA overestimated the results of determination of deoxynivalenol in beer by 363–697 % and with zearalenone there were found false positive results.
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