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Хемилюминесцентный метод определения микроорганизмов, основанный на определении аденозинтрифосфата (АТФ), который содержится в клетках бактерий, в целом состоит из трех стадий: пробоподготовки, устраняющей мешающее влияние матрицы образца, аналитической реакции и обработки полученного сигнала. Данная работа направлена на совершенствование второго этапа анализа – оптимизацию условий проведения хемилюминесцентной реакции. АТФ инициирует хемилюминесценцию люминола, интенсивность которой прямо пропорциональна концентрации АТФ. Нами были оптимизированы условия проведения данной хемилюминесцентной реакции для определения общего микробного числа (ОМЧ) в водных средах. Для этого проведено сравнение двух хемилюминесцентных реагентов: люминола и его гидрофобного аналога N-октиллюминола, который способен образовывать мицеллы. Переход к хемилюминесцентной системе на основе N-октиллюминола позволил увеличить чувствительность метода в 5.5 раз за счет микроконцентрирования аналита в ядре собственных мицелл. Нами было определено и обсуждено влияние неионогенных поверхностно-активных веществ на хемилюминесцентный способ определения бактерий. При этом выявлено, что добавление неионогенных поверхностно-активных веществ ухудшает аналитический сигнал за счет уменьшения локальной концентрации АТФ в мицеллах. Для аналитических систем, основанных на окислении люминола и N-октиллюминола, определены показатели точности, правильности, повторяемости и внутрилабораторной прецизионности. Применение разработанной хемилюминесцентной системы позволило существенно улучшить методику определения ОМЧ с переходом от качественного определения микроорганизмов к количественному измерению в диапазоне от 4×105 до 1×107 КОЕ/мл. При этом погрешность определения была снижена с 80 до 26 %., The chemiluminescent method for the determination of microorganisms based on the determination of adenosine triphosphate (ATP), which is contained in bacterial cells, generally consists of three stages - sample preparation eliminating the interfering effect of the sample matrix, analytical reaction and processing of the received signal. Current work is aimed at improving the second stage of the analysis - the optimization of the conditions for chemiluminescent reactions. ATP contained in bacteria initiates chemiluminescence of luminol, the intensity of which is directly proportional to the concentration of ATP. We have optimized the conditions for this chemiluminescent reaction to determine the total viable count (TVC) in aqueous media. Two chemiluminescent reagents were compared for this purpose: luminol and its hydrophobic analog N-octiluminol, which is capable of forming micelles. The introduction of N-octilluminol-based chemiluminescent system made it possible to increase the sensitivity of the method by a factor of 5.5 due to the microconcentration of the analyte in the nucleus of its own micelles. We have determined and discussed the effect of nonionic surfactants on the chemiluminescent method for determining the bacteria. It was found that the addition of nonionic surfactants degrades the analytical signal by reducing the local concentration of ATP in micelles. The accuracy, correctness, repeatability and intralaboratory precision were determined for the analytical systems based on the oxidation of luminol and N-octyluminol. The use of the developed chemiluminescent system allowed us to significantly improve the method for the determination of TVC with the transition from the qualitative determination of microorganisms to the quantitative measurement in the range of 4×105 to 1×107 CFU/ml. At the same time, the error was reduced from 80% down to 26%.

For the successful validation and verification of the HACCP system, a food business operator is obliged, among other duties, to have continuous microbiological data of carcasses which are followed by a certain dynamic that the subject himself prescribed. To obtain these data, it is necessary to perform systematic monitoring of indicator microorganisms. The most important meat hygiene indicators are Total Viable Counts (TVC) and Enterobacteriaceae (EC). TVC are defined as indicators of overall slaughter hygiene (equipment, environment, tools, workers), while EC are indicators of faecal contamination on carcasses. The aim of this study was to determine the microbial contamination of cattle and pig carcasses, level of hygiene of the slaughter process as well as whether variations hygiene levels were related to seasonality in Serbia during 2017. The year was divided into four quarters of three months each, while the microbiological results were classified into three levels of hygiene status (unsatisfactory, satisfactory or acceptable). The highest percentage of the results surveyed during the entire study was at a satisfactory hygiene level. Furthermore, we found there were differences in results between the quarters, which could be associated with seasonality. The best microbiological results, and so the best hygiene of carcasses, was recorded in the period April, May, June, while the worst microbiological results were observed in the period of July, August and September.

For the successful validation and verification of the HACCP system, a food business operator is obliged, among other duties, to have continuous microbiological data of carcasses which are followed by a certain dynamic that the subject himself prescribed. To obtain these data, it is necessary to perform systematic monitoring of indicator microorganisms. The most important meat hygiene indicators are Total Viable Counts (TVC) and Enterobacteriaceae (EC). TVC are defined as indicators of overall slaughter hygiene (equipment, environment, tools, workers), while EC are indicators of faecal contamination on carcasses. The aim of this study was to determine the microbial contamination of cattle and pig carcasses, level of hygiene of the slaughter process as well as whether variations hygiene levels were related to seasonality in Serbia during 2017. The year was divided into four quarters of three months each, while the microbiological results were classified into three levels of hygiene status (unsatisfactory, satisfactory or acceptable). The highest percentage of the results surveyed during the entire study was at a satisfactory hygiene level. Furthermore, we found there were differences in results between the quarters, which could be associated with seasonality. The best microbiological results, and so the best hygiene of carcasses, was recorded in the period April, May, June, while the worst microbiological results were observed in the period of July, August and September.

Processing of almond was done at different temperatures (80, 90, 100 and 110°C), to produce almond milk samples (A, B, C and D). The almond milk sample (C) processed at normal boiling temperature (100°C) kept as reference standard. Using different standard analytical methods, almond milk were analyzed for physiochemical, microbiological, and sensory attributes. Due to increase in temperature the crude fat and moisture content decreased significantly (p<0.05) from 3.40 to 3.14% and 86.05 to 83.78%, respectively; while significant increase (p<0.05) were observed in protein, ash, crude fiber and carbohydrate contents. The pH and Total solids of almond milk significantly increased from 6.75 to 6.84 and 13.89 to 15.11% respectively. Total viable count significantly decrease (p<0.05) from 2.24-1.33×103 CFU/ml for sample A to D processed at 80-110°C, while yeast and mold from 1.22-0.35×102 CFU/ml. The mean value score awarded to all sensory attributes increase from A to C but decrease in D. Almond milk products were acceptable, highest acceptability score (8.33) awarded to milk sample C processed at 100°C followed by samples B, A and D. Processing of almond milk at 100°C provide the better milk product with all measured characteristics suggested for almond milk processing.

Processing of almond was done at different temperatures (80, 90, 100 and 110°C), to produce almond milk samples (A, B, C and D). The almond milk sample (C) processed at normal boiling temperature (100°C) kept as reference standard. Using different standard analytical methods, almond milk were analyzed for physiochemical, microbiological, and sensory attributes. Due to increase in temperature the crude fat and moisture content decreased significantly (p<0.05) from 3.40 to 3.14% and 86.05 to 83.78%, respectively; while significant increase (p<0.05) were observed in protein, ash, crude fiber and carbohydrate contents. The pH and Total solids of almond milk significantly increased from 6.75 to 6.84 and 13.89 to 15.11% respectively. Total viable count significantly decrease (p<0.05) from 2.24-1.33×103 CFU/ml for sample A to D processed at 80-110°C, while yeast and mold from 1.22-0.35×102 CFU/ml. The mean value score awarded to all sensory attributes increase from A to C but decrease in D. Almond milk products were acceptable, highest acceptability score (8.33) awarded to milk sample C processed at 100°C followed by samples B, A and D. Processing of almond milk at 100°C provide the better milk product with all measured characteristics suggested for almond milk processing.

The aim of this study was to evaluate the effectiveness of different packaging conditions (vacuum and modified atmosphere) on the microbiological status (total viable count, lactic acid bacteria and Enterobacteriaceae), total volatile basic nitrogen (TVB-N) and pH in minced pork meat. Pork mince was packaged in vacuum, modified atmosphere with 20% O2, 50% CO2 and 30% N2 (MAP 1) or modified atmosphere with 20% O2, 30% CO2 and 50% N2 (MAP 2), refrigerated at 3±1°C and examined on the days 0, 3, 6, 9 and 12 of storage. The average total viable counts and total Enterobacteriaceae counts in vacuum packaged mince were statistically significantly higher (p lt 0.01; p lt 0.05) than in modified atmosphere packaged mince with both combinations of gases, on different days of storage. The largest decrease of total viable count and Enterobacteriaceae count was noted in modified atmosphere packaged mince with the higher concentration (50%) of CO2.

The aim of this study was to evaluate the effectiveness of different packaging conditions (vacuum and modified atmosphere) on the microbiological status (total viable count, lactic acid bacteria and Enterobacteriaceae), total volatile basic nitrogen (TVB-N) and pH in minced pork meat. Pork mince was packaged in vacuum, modified atmosphere with 20% O2, 50% CO2 and 30% N2 (MAP 1) or modified atmosphere with 20% O2, 30% CO2 and 50% N2 (MAP 2), refrigerated at 3±1°C and examined on the days 0, 3, 6, 9 and 12 of storage. The average total viable counts and total Enterobacteriaceae counts in vacuum packaged mince were statistically significantly higher (p lt 0.01; p lt 0.05) than in modified atmosphere packaged mince with both combinations of gases, on different days of storage. The largest decrease of total viable count and Enterobacteriaceae count was noted in modified atmosphere packaged mince with the higher concentration (50%) of CO2.

The aim of this study was to evaluate the effectiveness of different packaging conditions (vacuum and modified atmosphere) on the microbiological status (total viable count, lactic acid bacteria and Enterobacteriaceae), total volatile basic nitrogen (TVB-N) and pH in minced pork meat. Pork mince was packaged in vacuum, modified atmosphere with 20% O2, 50% CO2 and 30% N2 (MAP 1) or modified atmosphere with 20% O2, 30% CO2 and 50% N2 (MAP 2), refrigerated at 3±1°C and examined on the days 0, 3, 6, 9 and 12 of storage. The average total viable counts and total Enterobacteriaceae counts in vacuum packaged mince were statistically significantly higher (p lt 0.01; p lt 0.05) than in modified atmosphere packaged mince with both combinations of gases, on different days of storage. The largest decrease of total viable count and Enterobacteriaceae count was noted in modified atmosphere packaged mince with the higher concentration (50%) of CO2.

This thesis evaluates the MicroFoss system, also known as the BioSys or the SolerisÒ system, as a rapid method for microbiological analyses of eggs and egg products. Commercially assays for assessment of microbiological quality and sanitation conditions during processing, which are total viable counts (TVC), coliforms and Escherichia coli, are evaluated in comparison to the cultural methods. Concurrently, this thesis reports development of a new medium for the MicroFoss Salmonella detection, since this test is not available.Good correlations between the cultural TVCs and the MicroFoss detection times (DTs) were obtained (r ³-0.84) in all egg products. Preliminary results of a correlation between the cultural TVCs dominated by Pseudomonas fluorescens and the MicroFoss DTs suggested that the MicroFoss could be used for investigation of spoilage and shelf life prediction of eggs.High agreements (r ³-0.93) between the MicroFoss and cultural methods for coliforms and E. coli in eggs and egg products was achieved as well as the accuracy, sensitivity and specificity. The MicroFoss for estimations of TVC, coliforms and E. coli could be completed in less than 24h, compared to 48-72h required for the cultural methods. Hence, the MicroFoss could be used as a rapid screening for quality control of eggsand egg products.While a new propylene glycol-based selective medium developed was effective for detection of Salmonella, possible false positive results caused by certain bacteria, such as Enterobacter cloacae, E. coli, Klebsiella pneumoniae and Proteus mirabilis, were encountered. When this selective medium was supplemented with 40 mg/L of novobiocin, or 1 or 2 mL/L of Niaproof 4, these non-salmonellae were greatly suppressed or eliminated, particularly Pr. mirabilis, whilst this did not affect growth of Salmonella. Various serotypes of Salmonella including strains of atypical biotype were able to acidify propylene glycol in these media.Detection of Salmonella in naturally conta

Diplomová práce je zaměřena na ověření mikrobiální kontaminace ejakulátu. Zjišťovali jsme výskyt jednotlivých druhů a celkový počet mikroorganismů v 1 ml spermatu. Vyšetřili jsme 40 vzorků ejakulátu mužů evidovaných v Centru asistované reprodukce SANUS, s. r. o., v Pardubicích. Z jednotlivých vzorků jsme vykultivovali jeden aţ pět různých bakteriálních druhů. V ejakulátech se vyskytovaly Staphylococcus sp. - v 36 (90 %) vzorcích, koryneformní bakterie - v 31 (77,5 %), Streptococcus sp. - v 27 (67,5 %), Micrococcus sp. - ve 14 (35 %), Neisseria subflava - v 5 (12,5 %) a Lactobacillus sp. - ve 2 (5 %). Bakterie rodu Bacillus a Enterococcus, dále Facklamia hominis a Morganella morganii jsme prokázali vždy v 1 (2,5 %) vzorku. Celkové počty živých mikroorganismů (CFU) v 1 ml ejakulátu jsme zjišťovali metodou roztěru na krevní agar a ředicí metodou. Metodou roztěru na krevní agar jsme vyšetřili 40 vzorků ejakulátu. V 5 (12,5 %) vzorcích jsme zjistili 1,0x102 - 4,0x102 CFU/ml, v 9 (22,5 %) 1,0x103 - 9,6x103 CFU/ml, ve 13 (32,5 %) 1,1x104 - 8,0x104 CFU/ml, v 6 (15 %) 1,2x105 - 6,0x105 CFU/ml a v 7 (17,5 %) 106 CFU/ml a více. Ředicí metodou jsme vyšetřili 33 vzorků ejakulátu. Ve 2 (6,1 %) vzorcích jsme prokázali méně neţ 102 CFU/ml, ve 4 (12,1 %) 3,2x102 - 9,0x102 CFU/ml, v 15 (45,5 %) 1,3x103 - 9,0x103 CFU/ml, v 9 (27,3 %) 1,1x104 - 5,6x104 CFU/ml, ve 3 (9,1 %) 1,6x105 - 2,1x105 CFU/ml ejakulátu., The thesis is focused on the verification of microbial contamination of the ejaculate. We were investigating the occurrence of individual species and the total viable count of microorganisms in 1 ml of semen. We examined 40 semen samples of the men registered at the Centre for Assisted Reproduction SANUS, s. r. o., Pardubice. From each sample, we cultured from one to five different bacterial species. The ejaculates contained Staphylococcus sp. - in 36 (90 %) samples, coryneform bacteria - in 31 (77,5 %), Streptococcus sp. - in 27 (67,5 %), Micrococcus sp. - in 14 (35 %), Neisseria subflava - in 5 (12,5 %) and Lactobacillus sp. - in 2 (5 %). Bacteria of the genus Bacillus and Enterococcus, further Facklamia hominis and Morganella morganii are always found in 1 (2,5 %) sample. We investigated the total number of live microorganisms in 1 ml of ejaculate using the smear of blood agar and the dilution method. We examined 40 samples of semen using the smear of blood agar. In 5 (12,5 %) samples we found 1,0x102 - 4,0x102 CFU/ml, in 9 (22,5 %) 1,0x103 - 9,6x103 CFU/ml, in 13 (32,5 %) 1,1x104 - 8,0x104 CFU/ml, in 6 (15 %) 1,2x105 - 6,0x105 CFU/ml and in 7 (17,5 %) 106 CFU/ml or more. We examined 33 samples of semen using the dilution method. In 2 (6,1 %) samples we found less than 102 CFU/ml, in 4 (12,1 %) 3,2x102 - 9,0x102 CFU/ml, in 15 (45,5 %) 1,3x103 - 9,0x103 CFU/ml, in 9 (27,3 %) 1,1x104 - 5,6x104 CFU/ml and in 3 (9,1 %) 1,6x105 - 2,1x105 CFU/ml., Katedra biologických a biochemických věd, 1. Prezentace výsledků diplomové práce 2. Diskuze k posudkům vedoucího a oponenta diplomové práce 3. Diplomantka zodpověděla všechny dotazy a přípomínky k DP, Dokončená práce s úspěšnou obhajobou