Fresh foods, including meats and produce are the fastest growing market in the supermarket and the class of foods most likely to cause a bacterial foodborne illness. As the rate of consumption of perishable products increases, rapid detection of pathogens within the food supply becomes a critical issue. Current methods used for the detection of bacteria that cause food-borne illnesses are time consuming, expensive and often require selective enrichment. In this study we adapted a separation technique originally developed for PCR to extract bacteria from ground beef using β-cyclodextrin (β-CD) and milk protein coated activated carbon (MP-CAC) as filtration agents. The recovered bacteria were bound to a gold slide via a 3-mercaptophenylboronic acid (3-MPBA) sandwich assay and detected with Surface Enhanced Raman Spectroscopy (SERS). The 3-MPBA sandwich assay used with the separation technique allowed detection of Salmonella enterica Enteritidis (BAA-1045), separated from a ground beef matrix, as low as 1x102 CFU/g. Detection at this level was accomplished in less than 8 hours, significantly faster than plate count or enrichment methods that require multiple days. Previously, SERS has been used to detect bacteria within simple matrices; this is the first study to have utilized SERS bacterial detection in a ground beef.

Use of the bioluminometric ATP assay was evaluated for estimating total bacterial counts in ground beef. Minimum sensitivity was found to be 10⁶ cfu/g using a double filtration procedure for sample preparation. Although ATP content per cfu decreased approximately 10 fold during storage, correlation of total aerobic plate count (APC) with microbial ATP content was 0.96. Selective non-microbial ATP extraction with ATPase treatment was evaluated for use in conjunction with the double filtration procedure to increase assay sensitivity. The new method was effective for removing additional non-microbial ATP without reducing ATP in bacteria. Estimated APC values were generally accurate to within ±0.50 log for ground beef samples above the detection limit of 5 x 10⁴ cfu/g. ATPase treatment increased sensitivity of the ATP assay and APC estimation by about 1 log while increasing assay time by 40 minutes, for a total of 60 minutes for 4 samples assayed in triplicate. The ATP assay was evaluated for use with ground beef patties inoculated with mixed ground beef spoilage flora, <i>Pseudomonas</i>, or <i>Lactobacillus</i> and stored at 2°C or 10°C using oxygen permeable or impermeable (vacuum) packaging. Excellent correlation (r²=0.95) was obtained for each inoculum and storage condition over the range of 5 x 10⁴ to 1 x 10⁹ cfu/g, when estimated APC values were compared with experimentally observed APC values. Usefulness of the ATP assay for estimating APC values of frozen ground beef was evaluated. Retail ground beef and <i>Lactobacillus</i>- and <i>Pseudomonas</i>-inoculated beef were frozen and thawed at different rates and examined for APC and microbial ATP content. Results indicated that, although freezing and thawing lowered numbers of <i>Pseudomonas</i>, APC values and microbial ATP content closely correlated. APC estimates were generally accurate to within 1/2 log. The importance of using an ATP assay standard to correct for variable enzyme activity and presence of quenching factors was demonstrated, and improved formulae were developed for optimum assay standard use. Alternate regression methods were evaluated for estimation of APC values but did not yield enhanced accuracy. Only one regression equation was needed for estimating APC values of ground beef containing different types of bacteria stored in various ways. Therefore, little knowledge of ground beef history is needed in order to rapidly and accurately estimate microbial numbers in ground beef using the bioluminometric ATP assay. / Ph. D.

The seed germination process during sprout production provides suitable environmental conditions for the growth of pathogenic bacteria, such as Listeria monocytogenes. A potential way to control this bacterial growth is through the use of bacteriophages, which are naturally occurring viruses that specifically attack bacterial targets and have been shown to be effective antimicrobials in some foods. Therefore, the objective of this study was to evaluate the antimicrobial susceptibility of L. monocytogenes to bacteriophage on alfalfa sprouts during seed germination and subsequent refrigerated storage at 4 °C. Alfalfa sprout seeds were dip-inoculated with 5.5 x 105 CFU/ml L. monocytogenes serogroups 1 and 4. This was followed by treatment with the commercial bacteriophage LISTEX™ P100 at a concentration of 5.3 x 107 PFU/ml. The seeds were then soaked and germinated for 80 h using the glass jar method. The concentration of L. monocytogenes was determined every 24 h using PALCAM agar plated in triplicate. When compared to the spiked, untreated control, treatment of sprout seeds with LISTEX™ P100 resulted in a statistically significant (p < 0.05) reduction of 1.6 log10 CFU/g L. monocytogenes after the initial 24 h of germination. However, the bacteriophage did not show a lasting inhibitory effect, with no statistically significant reductions in L. monocytogenes growth as compared to the control at subsequent time points. The bacteriophage remained stable over the entire germination and storage period. Although biocontrol of Listeria with bacteriophages has high potential to serve as an alternative strategy to control foodborne illnesses, factors such as phage delivery and dose optimization in sprouts need to be further investigated.

The primary aim of this investigation was to identify and assess the interactions (synergies and antagonisms) that exist between 20 minute detergent and 5 minute disinfectant treatments upon three factory isolated strains of surface adhered (1-hour attached) and surface adapted (24-hour biofilm) populations of Escherichia coli and Listeria monocytogenes, plus a comparison with vero-toxin producing strains of E. coli, when used as part of a cleaning and disinfection regime. The detergents chosen for assessment were two non-ionic (91/4 - Alcohol Ethoxylate and KCL5 - Polyethoxylated Alcohol), two anionic (LX28 - Sodium Lauryl Sulphate and Nec28 - Sodium Laurylether Sulphate) and two novel bismuth thiols (BisEDT - 1:1 Bismuth nitrate 1,2-ethanedithiol and BisTOL - 2:1 Bismuth nitrate 3,4-dimercaptotoluene), developed at Winthrop University Hospital, New York. The disinfectants chosen for assessment were a quaternary ammonium compound (BAC - Benzyl alkonium Chloride) and a chlorine releasing agent (NaDCC - Sodium Dichloroisocyanurate). The investigation showed that there were no specific cleaning and disinfection regimes that will adequately target both E. coli and L. monocytogenes strains. It was also concluded that to maximise the removal and disinfection of persistent strains of a given microorganism, it may be necessary to design a regime to specifically target not just the species, but the strain involved and where possible requires mechanical cleaning. The novel bismuth thiols were seen to be promising detergents to aid in the removal of E. coli strains and warrant further attention for future studies. Finally, an investigation to identify possible mechanisms of resistance to disinfectant treatments following detergent treatment, showed that different detergents can induce expression of the stress response proteins, HSP60 and HSP70, at differing levels of expression after the same contact time and against different states of adherent populations, i.e. 1-hour attached or 24-hour biofilm populations.

There is demand for improved security of refrigerated ready-to-eat meats. Antimicrobial edible films and coatings could function as an added barrier against post-processing contamination. Hops and hop extracts are known for their antimicrobial efficacy which is attributed to key antimicrobial components including humulones, lupulones, xanthohumol and various terpenoids. Yet, hop ethanol extract has not been studied as an antimicrobial to incorporate into edible protein films and/or coatings. The overall objective of this research was to evaluate hop ethanol extract as an antimicrobial agent incorporated into edible soy protein isolate (SPI) films and coatings, and the influence on the shelf-life and sensory attributes of bologna. Hop ethanol extract was examined for minimum inhibitory concentration before the extract was incorporated into a 6% SPI solution at 0, 10, and 20% levels to determine antimicrobial efficacy as a cast film and simulated coating via zone of inhibition against Listeria monocytogenes strains ATCC 4644, UKADL and ATCC 49594. The results showed that hop ethanol extract alone was inhibitory of all three strains. Moreover, the hop ethanol extract, when incorporated at 10 and 20% (v/v) into edible soy protein isolate (SPI) films and simulated coatings, exhibited antimicrobial action against all three L. monocytogenes strains. Key antimicrobial components, as mentioned above, were identified in the hop ethanol extract via mass spectrometry. The SPI with 10% incorporated hop ethanol extract (SPI+10%hop) antimicrobial coating was applied to bologna, prepared in lab without L. monocytogenes inhibitors, where it exhibited a significant (P ≤ 0.05) bacteriostatic effect against strain ATCC 4644. The SPI+10% hop coating was then applied to a commercial bologna to examine effects on shelf-life and sensory attributes. Significant differences (P ≤ 0.05) were found in instrumental red and yellow colors, however not in sensory color. There was no significant difference (P > 0.05) found in measured lipid oxidation between the bologna with no coating, SPI coating or SPI+10%hop coating. The incorporation of hop did exhibit a slightly bitter taste. Overall, these findings indicate that the SPI+10%hop antimicrobial coating functioned as an inhibitor of L. monocytogenes while producing minimal effects on shelf-life and sensory attributes of bologna.

No description available.

The research within this thesis sought to determine the ability of various animal derived fats and plant derived oils to support the survival of several pathogenic cocktails over a multitude of storage times. The Salmonella study explored the survival rate of a four strain Salmonella cocktail in beef tallow, pig lard, duck fat, coconut oil, and extra virgin olive oil over seven days at 26˚C and 37˚C storage. The animal fats and the coconut oil supported the survival of the bacteria until the conclusion of the study. The Shiga-toxin producing Escherichia coli study explored the survival rate of a five strain STECs cocktail in extra virgin olive oil over seven days at 26˚C and 37˚C storage. The two Listeria studies explored the survival rate of a four strain Listeria monocytogenes cocktail in extra virgin olive oil over several time periods with different frequencies of sample mixing. In vitro, all genuses showed a 2.5-log cfu/mL to ≥ 7-log cfu/mL reduction in the extra virgin olive oil by the conclusion of the experiments. Extra virgin olive oil was then applied to cooked pork tenderloin, cheddar cheese snack squares, and turkey lunchmeat in hopes of inhibiting the L. monocytogenes cocktail. No reduction was observed.

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.

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.

Com a finalidade de se avaliar a correlação entre o sistema SimPlate e metodologias convencionais na análise microbiológica de alimentos, dezoito alimentos (nove de origem vegetal e nove de origem animal) foram analisados para contagem total de mesófilos aeróbios, bolores e leveduras e coliformes totais e fecais. Os resultados das análises microbiológicas foram comparados através do teste F, tendo havido diferença significativa (p<0,05) entre os métodos. As médias foram submetidas ao teste t-Student (p<0,05); para a contagem total de mesófilos aeróbios apenas em dois dos quinze alimentos considerados, maçã e beterraba, as metodologias convencional e SimPlate não diferiram entre si; para bolores e leveduras as metodologias não diferiram em quatro dos treze alimentos considerados, lingüiça, peixe, cheiro-verde e beterraba, e para coliformes totais, em seis dos doze alimentos considerados, carne suína, frango, lingüiça, leite cru, pêra e tomate. Uma análise de regressão dos dados mostrou boa correlação entre os dados obtidos para coliformes totais, pela metodologia dos tubos múltiplos e o SimPlate (0,88) e os dados de mesófilos aeróbios, obtidos pelo PCA e o SimPlate (0,80) e baixa correlação entre os dados obtidos na determinação de bolores e leveduras pelo BDA e o SimPlate (0,69). / In order to evaluate correlation among SimPlate system and conventional methods for food microbiological analysis, eighteen foods (nine from vegetable origin and nine from animal origin) were analyzed for mesophilic aerobic, yeasts and molds, total coliforms and fecal coliforms. Results of the three analysis were compared and significant differences (p<0,05) occurred among the majority of foods. For mesophilic aerobic counts, between the methodologies, PCA and SimPlate, difference were not significant in two of fifteen considered foods, apple and beetroot; for yeasts and molds, using acidify PDA and SimPlate, difference were not significant in four of thirteen considered foods, mixed sausage, fish, parsley and Welsh onion and beetroot; for total coliforms, using three-tube method and SimPlate, difference were not significant in six of twelve considered foods, raw pork, chicken, mixed sausage, raw milk, pear and tomato. Regression analysis of data showed correlation coefficients like, 0,88 for three-tube method and SimPlate; 0,80 between PCA and SimPlate and 0,69, between acidify PDA and SimPlate.