Community-acquired Clostridium difficile infections have recently been increasing in incidence and severity. Several studies have isolated C. difficile spores from livestock and retail meats, suggesting that food may play a role in transmission. No research has been done, however, on what food conditions might allow for the survival and/or growth of the bacterium. We therefore modelled the minimum thresholds for C. difficile growth under low pH, water activity (aw), and temperature. We also sampled retail ground meats, cheese, and milk for the presence of C. difficile spores and subtyped food isolates for comparison with clinical strains. We found that C. difficile growth could be prevented by refrigeration temperatures. C. difficile spores were also detected for the first time in Canada in ground lamb, ground turkey, ground chicken, cheese and milk. The majority of these food isolates were genetically similar to epidemic strain NAP7/078, suggesting that food may not be a direct vector for C. difficile transmission, but could still be clinically relevant.

A presença de Salmonella em 200 amostras de alimentos crus de origem animal foi investigada empregando-se os dois ensaios imunoenzimáticos rápidos TECRA™ Salmonella VIA e TECRA™ Salmonella UNIQUE (TECRA Diagnostics, Rosewille, NSW, Australia) e o método de cultura convencional empregado rotineiramente no Instituto Adolfo Lutz, São Paulo, SP. Quarenta e cinco amostras (22.5%) foram Salmonella positivas por pelo menos um dos três métodos. O número de amostras positivas de acordo com o método analítico foi 34 (75,6%) para o método de cultura convencional, 29 (64,4%) para TECRA™ Salmonella VIA e 27 (60.0%) para TECRA™ Salmonella UNIQUE. O método de cultura convencional detectou quatro amostras positivas não detectadas por nenhum dos outros dois métodos rápidos. TECRA™ Salmonella UNIQUE detectou sete amostras positivas não detectadas pelos demais métodos. Uma amostra foi positiva apenas pelo método TECRA™ Salmonella VIA. Considerando todos os resultados (positivos e negativos) o teste de qui quadrado de McNemar indicou que as diferenças entre os resultados obtidos pelos métodos rápidos, quando comparados aos obtidos pelo método convencional, não foram estatisticamente significativas (p>0.05). / The presence of Salmonella in 200 raw food samples of animal origin was investigated by means of rapid immunoassays TECRA™ Salmonella VIA and TECRA™ Salmonella UNIQUE (TECRA Diagnostics, Rosewille, NSW, Australia) and the cultural procedure used routinely in Instituto Adolfo Lutz, Sao Paulo, SP. Forty-five samples (22.5%) were Salmonella positive by at least one of the three methods. The number of positive samples according to the analytical method was 34 (75.6%) for the cultural procedure, 29 (64.4%) for TECRA™ Salmonella VIA and 27 (60.0%) for TECRA™ Salmonella UNIQUE. The cultural method detected four positive samples that both rapid methods were unable to detect. TECRA™ Salmonella UNIQUE detected seven positive samples that were not detected by the two other methods. One sample was positive by the TECRA™ Salmonella VIA exclusively. Considering overall results (positive and negative) McNemar\'s chi square tests indicated that the differences between results given by the rapid immunoassays when compared to those of the cultural method were not significant (p>0.05).

A preocupação constante com a inocuidade e a qualidade dos alimentos irradiados, aliada à tendência de se estudar os aspectos sensoriais desses alimentos, foram a motivação para o desenvolvimento deste trabalho que teve como objetivos verificar os efeitos da radiação ionizante produzida pelo 60Co na sobrevivência de Salmonella sp em hambúrgueres de carne de frango congelados e verificar a aceitação do produto irradiado durante o seu período de estocagem. Amostras de hambúrguer de carne de frango congelados, inoculados com quatro cepas diferentes de Salmonella sp foram expostos a níveis de radiação com doses variando entre O e 3,5 kGy para verificação da sobrevivência da população de Salmonella sp presente. Amostras não inoculadas e irradiadas com doses de 0,5 e 7 kGy foram submetidas à avaliação sensorial por painel não treinado, pelo período de 120 dias, com o intuito de se verificar o decaimento da qualidade do produto. Os valores D10 encontrados para Salmonella sp variaram entre 1,02 e 1,32 kGy, portanto, as doses de 5 e 7 kGy, utilizadas nas fases seguintes do experimento, seriam suficientes para reduzir a população de Salmonella sp no produto em 5-6 ciclos logarítmicos. A exposição às doses de 5 e 7 kGy não afetou as características sensoriais do produto. A vida de prateleira do hambúrguer de carne de frango congelado e irradiado com doses de 5 e 7 kGy foi de, no mínimo, 120 dias, ou seja, igual à do produto não irradiado. / Safety and quality of irradiated foods are still of great concern to researchers and consumers, in general. The aims of this research were to study the effects of ionizing radiation from 60Co on the population of Salmonella sp inoculated in frozen chicken meat patties as well as on their sensory characterisitcs during the storage period. Samples of frozen chicken meat patties, inoculated with a pool of four strains of Salmonella sp, were exposed to irradiation doses varying from 0 to 3.5 kGy in order to determine their sensitivity (D10). Non inoculated samples of frozen chicken meat patties exposed to 5 and 7 kGy and a non inoculated and non irradiated sample were submitted to sensory evaluation by a non-trained panel. 010 values varied from 1.02 to 1.32 kGy Samples exposed to 5 and 7 kGy did not showed appreciable changes during the storage period of 120 days. Therefore, irradiation can be applied to chicken meat patties in order to improve their safety during a storage period of, at least, 120 days.

Clostridium perfringens é um dos microrganismos mais freqüentemente envolvidos em surtos de enfermidades transmitidas por alimentos. Este microrganismo pode ser classificado em cinco tipos toxigênicos (A-E), de acordo com a detecção dos genes codificadores de suas principais toxinas: alfa (cpa), beta (cpb), épsilon (etx) e iota (iap), sendo que técnicas moleculares empregando a PCR são atualmente utilizadas para genotipagem desses isolados. Alguns isolados de C. perfringens produzem uma enterotoxina (CPE) que é responsável pelos sintomas clínicos desenvolvidos em casos de toxinfecção alimentar, sendo que esta toxina é codificada pelo gene cpe. A simples detecção de C. perfringens em um alimento, mesmo naqueles suspeitos de causar surtos, não é suficiente para considerá-lo como de risco à saúde do consumidor. Isto porque dentre os isolados de C. perfringens apenas um número muito pequeno apresenta o gene cpe. Além disso, isolados de C. perfringens não produtores de CPE estão amplamente disseminados no ambiente, em alimentos e mesmo em fezes de pessoas. Desta forma, com o presente estudo verificou-se a freqüência de C. perfringens dentre isolados de clostrídios sulfito redutores, a freqüência de C. perfringens potencialmente enterotoxigênicos e sua variabilidade genética, de modo a evidenciar a importância dessas cepas como causadoras de doenças, além de fornecer subsídios para melhorar os conhecimentos sobre as características das cepas circulantes em nosso meio. Foram utilizados 335 isolados de clostrídios sulfito redutores provenientes de alimentos (126), solo (84) e fezes de animais (125). Dos 335 isolados, 146 (43,6%) foram caracterizados, através de reações bioquímicas e moleculares, comoC. perfringens, sendo 75 isolados (59,5%) provenientes de alimentos, 43 (51,2%) de solo e 28 (22,4%) de fezes de animais. Todas as cepas de C. perfringens analisadas foram tipadas como C. perfringens tipo A. Dos 75 isolados de C. perfringens provenientes de alimentos, 20 apresentaram o gene cpe, sendo 13 (65%) com localização cromossomal; nas demais cepas não foi possível determinar sua localização. Nos isolados de C. perfringens provenientes de solo e das fezes de animais não se verificou a presença desse gene. Das 20 cepas de C. perfringens que apresentaram o gene cpe detectou-se em 15 a produção de enterotoxina; as cinco cepas restantes não apresentaram esporulação no meio DUNCAN STRONG modificado, não sendo possível avaliar sua atividade enterotoxigênica. As 146 cepas de C. perfringens quando submetidas à PFGE geraram 69 perfis PFGE distintos, sendo 42 exclusivos para uma única cepa, indicando uma grande variabilidade genética, entre isolados provenientes de amostra de alimentos, fezes ou solo. A utilização de clostrídios sulfito redutores, ou mesmo de C. perfringens como indicador de possível risco à saúde dos consumidores pode levar à condenação desnecessária de alimentos, uma vez que existe baixa correlação entre costrídios sulfito redutores e C. perfringens, independente da fonte de isolamento, além da baixa freqüência do gene cpe nas cepas estudadas. / Clostridium perfringens is one of the most frequently microorganism involved in outbreaks of foodborne diseases. This microorganism can be classified into five toxigenic types (A to E), according to the detection of genes encoding its major toxins: alpha (cpa), beta (cpb), epsilon (etx) and iota (iap). Molecular techniques using PCR are currently used for genotyping this isolates. Besides the major toxins, some isolates of C. perfringens produce an enterotoxin (CPE) that is responsible for clinical symptoms developed in cases of food poisoning. This enterotoxin is encoded by the cpe gene. The simple detection of C. perfringens in food, even in those suspected of causing outbreaks, is not enough to consider it as a risk to consumers´ health. This happens because among the isolates ofC. perfringens only a very small number shows the cpe gene. In addition, isolates of C. perfringens that do not produce CPE are widespread in the environment, food and even in feces of humans. Thus, the present study examined the frequency of C. perfringens isolates among sulfite reducing clostridia, the frequency of potentially enterotoxigenic C. perfringens and its genetic variability in order to highlight the importance of these strains in causing diseases, and provides subsidies to improve the knowledge about the strains that are circulating in our environment. A total of 335 isolates of sulfite reducing clostridia from foods (126), soil (84) and animal feces (125) were used. Among the 335 isolates, 146 (43.6%) were characterized by biochemical and molecular reactions as C. perfringens, being 75 (59.5%) from foods, 43 (51.2%) from soil and 28 (22.4%) from animal feces. All strains of C. perfringens were typed as C. perfringens type A. Of the 75 isolates of C. perfringens from food, 20 had the cpe gene, and in 13 (65%) the gene was chromosomally located. In the other strains it was not possible to determine the location of this gene. In isolates of C. perfringens from soil and animal feces the cpe gene was not present. Amongst the 20 strains of C. perfringens positive for cpe, enterotoxin production was detected in 15. Five strains showed no sporulation in the medium modified Duncan Strong, being not possible to verify their enterotoxigenic activity. All C. perfringens were subjected to PFGE and generated 69 different PFGE profiles, being 42 unique to a single strain, indicating a great genetic variability among isolates from food, feces or soil. The use of sulfite reducing clostridia, or even C. perfringens as an indicator of possible health risk to consumers can lead to unnecessary condemnation of food, since there is low correlation between sulfite reducing clostridia and C. perfringens, regardless of source of isolation. This study also shows a low frequency of cpe gene in the strains indicating the low risk in causing foodborne disease.

The house fly, Musca domestica can transmit human pathogens including Escherichia coli O157:H7 through regurgitation of ingested bacteria from the crop which is a foregut organ of house fly and stores the excess ingested nutrients. Interactions between the ingested bacteria and the crop have a direct influence on bacteria persistence, survival and ultimately fly vector competence. In this research, in situ crop vessel assay was developed to investigate bacterial growth within fly crops up to 48 hours post-ingestion. Flies were fasted for 12 h prior to feeding E. coli O157:H7 pEGFP and then fed bacteria with red food color which was added to confirm that flies had consumed the bacteria. After feeding, flies with red abdomens were aseptically dissected and crops were removed and maintained in sterile phosphate buffered saline in microtiter plates held at 32˚C. For each time point (0, 24 and 48 hours post-ingestion), five crops were homogenized individually using a tissue grinder and bacterial levels (CFU/crop) were monitored using plate counts. Confocal microscopy of intact crops was used to monitor biofilm development. There was no statistical difference in cell numbers (CFU/crop) over the 48 h incubation period. Microscopy showed that upon prolonged incubation, GFP-expressing E. coli within the crop produced biofilms. This method showed greater reproducibility in studying crop bacteria level than using a live fly feeding study. But this system was not recommended to study the interaction between bacteria and the crop of housefly.

An effective and rapid method for the separation of bacteria from food matrix remains a bottleneck for rapid bacteria detection for food safety. Bacteria can strongly attach to the food surface or internalize within the matrix which makes their isolation extremely difficult. Traditional methods of separating bacteria from foods routinely involve stomaching, blending and shaking, however these methods may not be efficient at removing all the bacteria from complex matrices. Here, we investigate the benefits of using enzyme digestion followed by immunomagnetic separation to isolate Salmonella from spinach and lettuce. Enzymatic digestion using pectinase and cellulase was able to break down the structure of the leafy green vegetables resulting in the detachment and release of Salmonella from the leaves. Immunomagnetic separation of Salmonella from the liquefied sample allowed an additional separation step to achieve a more pure sample without leaves debris that may benefit additional downstream applications. We have investigated the optimal combination of pectinase and cellulase for the digestion of spinach and lettuce to improve sample detection yields. The concentrations of enzymes used to digest the leaves were confirmed to have no significant effect on the viability of the inoculated Salmonella. Results reported that the recovery of the Salmonella from the produce after enzyme digestion of the leaves was significantly higher (P

The objective of this research was to study the impact of ripening inhibitor level and type on the formation, stability, and activity of antimicrobial thyme oil nanoemulsions formed by spontaneous emulsification. Oil-in-water antimicrobial nanoemulsions (10 wt%) were formed by titrating a mixture of essential oil, ripening inhibitor, and surfactant (Tween 80) into 5mM sodium citrate buffer (pH 3.5). Stable nanoemulsions containing small droplets (d < 70 nm) were formed. The antimicrobial activity of the nanoemulsions decreased with increasing ripening inhibitor concentration, which was attributed to a reduction in the amount of hydrophobic antimicrobial constituents transferred to the separated hydrophobic domain, mimicking bacterial cell membranes, by using dialysis and chromatography. The antimicrobial activity of the nanoemulsions also depended on the nature of the ripening inhibitor used: palm ≈ corn > canola > coconut which also depended on their ability to transfer hydrophobic antimicrobial constituents to the separated hydrophobic domain.

Temperature and relative humidity of produce throughout the cold chain can greatly affect the quality and safety of the food product. Different packaging systems or containers can provide better cooling environments for food products that could decrease temperature abuse and ultimately safety risks. In this study we compiled temperature and relative humidity profiles of broccoli packed in different shipping containers throughout the produce supply chain. The shipping containers looked at were the wax corrugated box, reusable plastic containers (RPC), and Eco Pack Green Box with and without a lid. Large differences were seen in the temperature profiles of each package during the first 15 hours of the cold chain with the wax-corrugated boxes showing the slowest rate of cooling. Growth rates of Salmonella sp. and Listeria monocytogeneson broccoli at different temperatures were also determined. Salmonella sp.showed a greater ability to grow on inoculated broccoli than Listeria monocytogenesduring higher temperatures such as 20° C and 37° C. Temperature profiles along with microbial counts from produce lots have been previously recorded; this is the first study to record temperature and relative humidity profiles in conjunction with bacterial growth data of lab inoculated produce.

The thesis contains studying material – textbook for the practice in food microbiology for the students of the secondary technical schools of chemistry and food science. Verification of practical utility and didactical advisability of submited exercices was realized at The Secondary Technical School of Chemistry in Brno.

In addition to proving an excellent source of nutrients, eggs are used in the food, cosmetic, and biotechnology industries for their rheological and bioactive properties. Much of the potential for the added value is in individual components of the egg, rather than the whole egg. At low speed centrifugation, yolk separates into two distinct fractions—granules and plasma. It is becoming increasingly popular in the industry to remove the plasma fraction of the egg yolk to use for its livetins, particularly immunoglobulin Y, leaving behind a granule by-product (“yellow cake”). Previous research has shown potential added-value from the granule fraction, especially from its phosvitin and phospholipids. Granules are protein aggregates with complexes of phosvitin and high density lipoproteins linked by phosphocalcic bridges. In their native form, the proteins are mostly insoluble, however previous studies have shown the links can be broken by alterations in pH, ionic strength, and mechanical treatments. This thesis project seeks to find potential uses for the egg yolk by product after the removal of the livetin fraction by means of further fractionation with mechanical treatment (filtration). Two variables were tested to extract more proteins from the yellow cake. Salt was added to 10% solids solution of yellow cake in water before filtration at four different NaCl levels: 0%, .05%, 1%, and 2.5%. Additionally pH was tested at four different levels: 4.6, 4.8, 5.0, 5.2. The samples were also tested for antibacterial properties against Escherichia v coli with a minimum inhibitory concentration assay (MIC). Analysis with BCA showed salt concentration had a significant effect on the yield of protein. The highest concentration of salt tested, 2.5%, had the highest protein yield. Additionally, SDS PAGE showed 2.5% salt had the most unique protein bands. This could be to the disruption of the phosphocalcic links between the phosvitin and HDL by NaCl, allowing the protein to solubilize. pH did not have a significant effect on the yield or types of proteins in the range tested in this experiment. There is no conclusive evidence of antibacterial properties against E. coli from the protein extract. The MIC assay had growth show up in all wells with the protein extract, however there was a visible decrease in turbidity with higher concentration of the protein extract. This could mean that the protein extract does have some antibacterial properties, but needs testing at higher concentrations or with isolated proteins/peptides. The SDS-PAGE revealed bands showing phosvitin present, which has known antibacterial properties. Overall, improvements to the methods for further protein extraction from egg yolk by-products will help lead the industry to finding novel uses and product applications.