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Salmonella enterica has been associated with several outbreaks due to consumption of low water activity foods including spices. Consequently, to improve microbiological quality, spices are commonly treated before ultimately reaching consumers. These processes may result in sub-lethal injury to cells, which can lead to an underestimation of microbial populations when plating on selective media. Sub-lethally injured cells are difficult to enumerate due to reduced ability to grow on selective microbiological media. Poor recovery of sub-lethally-injured cells may obscure process validation results and lead to overestimation of process effect. Therefore, this work was performed to determine the influence of agar overlay and media supplementation methods on the recovery of Salmonella from steam and ethylene oxide treated peppercorns and cumin seeds. Traditional agar overlay allowed recovery of significantly more Salmonella (p < 0.05) from inoculated peppercorns treated with steam (65.5C, 15 sec.) than selective media (XLT4) or solid agar overlay. Additional supplementation of the TSA base to contain 3,3'-thiodipropionic acid further improved Salmonella recovery from steam treated peppercorns (p < 0.05). For peppercorns and cumin seeds subjected to ethylene oxide, neither sodium pyruvate + yeast extract, 3,3'-thiodipropionic acid, glycerophosphate, ATP, nor magnesium enhanced recovery compared to overlay alone but both methods recovered significantly higher numbers than XLT-4 alone (p < 0.05) for both cumin seeds and peppercorns. / Master of Science in Life Sciences

Salmonella contamination of spices and other low water activity foods is a growing concern for the food industry due to increased frequency of salmonellosis outbreaks and detection-based product recalls. The impact of inoculation preparation on the survival of a Salmonella enterica and its proposed surrogate, Enterococcus faecium NRRL B-2385, on the whole black peppercorns and cumin seeds was examined. Three liquid inoculation methods (biofilm-inclusion, agar-grown, broth-grown) for Salmonella enterica and surrogate Enterococcus faecium and one dry transfer method for Salmonella enterica were developed then applied to whole peppercorn and cumin seeds. Spices were returned to original water activity (aw 0.3) and stored for 28 days with periodic sampling (0, 1, 7, 14, 21, 28 days) and surviving bacteria enumerated. Average log reductions (LR) over time were statistically analyzed to determine differences in stability during storage. Inoculation preparation was associated with significant differences in recovered Salmonella and Enterococcus from both peppercorn and cumin over the storage period. At 28 days, the most stable inoculations of Salmonella resulted from the biofilm-inclusion (-0.04 CFU/g LR) and agar grown (-0.75 CFU/g LR) methods on peppercorn and the biofilm inclusion method (-0.28 CFU/g LR) on cumin. Log reductions of Enterococcus faecium (-0.02 CFU/g LR biofilm-inclusion-peppercorn, -0.19 CFU/g LR agar-grown-peppercorn, -0.61 CFU/g LR biofilm-inclusion-cumin) were comparable to Salmonella after 28d desiccated storage. These results will guide the inoculation strategies for validating inactivation processes for reducing Salmonella on whole spices, and for comparisons of inactivation of Salmonella and its proposed surrogate Enterococcus faecium. / Master of Science in Life Sciences

Made available in DSpace on 2014-06-11T19:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-25Bitstream added on 2014-06-13T20:56:19Z : No. of bitstreams: 1 arakaki_rlm_me_sjrp.pdf: 890259 bytes, checksum: 8d0052a8a32ab44243e80d491899d7be (MD5) / Os basidiomicetos são conhecidos por sua capacidade em degradar a madeira. Essa característica deve-se à sua habilidade em oxidar a lignina presente nas plantas. Essa molécula é um composto recalcitrante formado por sub-unidades de fenilpropanóides e as enzimas capazes de degradá-la são conhecidas conjuntamente como enzimas ligninolíticas. Tais enzimas são inespecíficas. Essa característica confere-lhes o seu emprego na oxidação de compostos que possuem estrutura molecular semelhante à da lignina, tais como pesticidas e hidrocarbonetos poli-aromáticos, que possuem anéis aromáticos em sua estrutura. Dessa forma, é possível utilizar tais enzimas na degradação desses xenobióticos. Alguns basidiomicetos possuem a habilidade de crescer em meio com baixa atividade de água. Assim, é possível usá-los em processos de biorremediação de ambientes que possuem uma alta concentração de solutos, como em meios com alta salinidade. Esse trabalho teve como objetivo o estudo da capacidade de crescimento em meio líquido agitado com baixa atividade de água do basidiomiceto Dacryopinax elegans SXS323, promovida pela adição de cloreto de sódio 0,5 M, manitol 0,5 M ou glicerol 0,5 M ao meio de cultura; a produção de enzimas ligninolíticas e o seu poder de degradação do herbicida diurom nessas condições. Verificou-se que o basidiomiceto foi capaz de crescer sob todos os meios testados, sendo este mais pronunciado quando manitol ou glicerol foram acrescentados ao meio, averiguando-se um estímulo de crescimento. O manitol e o glicerol serviram como fonte secundária de carbono. O cloreto de sódio inibiu seu crescimento. Outro fator de inibição do crescimento foi a adição de diurom. A produção de enzimas ligninolíticas foi muito baixa, com pico de atividade de 3,67 U . L -1 com 144 horas de cultivo em meio com glicerol 0,5M. Apesar disso, detectou-se degradação... / The basidiomycetes are known for their ability to degrade wood. This feature is due to its ability to oxidize lignin of plants. This molecule is a compound formed by recalcitrant sub-units of phenylpropanoids and enzymes able to degrade it and are known as ligninolytic enzymes. These enzymes are nonspecific. This characteristic provides them the capability of oxidation of compounds that have molecular structures similar to lignin, such as pesticides and polyaromatic hydrocarbons, which possess aromatic rings in their structure. Thus, it is possible to use such enzymes in the degradation of xenobiotics. Some basidiomycetes have their ability to grow in media with low water activity. Thus, it can be used in bioremediation process in environments that have a high concentration of solutes, such as hypersaline environments. This work aimed to study the capability of the basidiomycete Dacryopinax elegans SXS323 to grow in liquid shaken with low water activity, promoted by the addition of sodium chloride 0.5 M, mannitol 0.5 M or glycerol 0.5 M to the medium culture, the production of ligninolytic enzymes and their degradation capability of the herbicide diuron in these conditions. It was found that the basidiomycete was able to grow in all media tested. The growth was most pronounced when mannitol or glycerol was added to the medium, probably working as a stimulus for growth, and were used as a secondary source of carbon. Sodium chloride inhibited its growth. Another factor inhibiting the growth was the addition of diuron. The ligninolytic enzyme production was very low, with peak activity of 3.67 U. L -1 with 144 hours in medium with glycerol 0.5 M. Nevertheless, we detected the degradation of the herbicide, registering 32.63% (glycerol 0.5 M, 192 hours), 25.47% (mannitol 0.5 M, 192 hours) 20.51% of reduction in medium with glucose 5.0 g . L -1 (240 hours)... (Complete abstract click electronic access below)

The pathogenic microorganism Salmonella enterica has been associated with several outbreaks and recalls of spices, herbs, and seeds. To control these pathogens additional treatment methods, such as fumigation with chlorine dioxide (ClO2) or hydrogen peroxide (H2O2) gas and recovery methods are needed. Recovery methods should accurately quantify all viable cells, even those injured, to prevent overestimation of treatment effectiveness. This study was performed to determine the effect of different recovery media and supplements on the recovery of multiple strains of S. enterica and Enterococcus faecium NRRL B2354, from chlorine dioxide or hydrogen peroxide treated low moisture foods (LMF) black peppercorns, dried basil leaves, and chia seeds. Also, this study aimed to compare the log reduction of these two microorganisms to evaluate E. faecium NRRL B2354 as a surrogate for S. enterica. On average, recovery of S. enterica was 3.43 log and 4.77 log CFU/g from ClO2 and H2O2 treated LMFs, respectively on the selective media Xylose Lysine Deoxycholate agar, while the average recovery on non-selective media was 4.50 log CFU/g and 5.74 log CFU/g from ClO2 and H2O2 treated LMFs, respectively. The use of non-selective media was correlated with increased recovery compared to selective media. In further studies, addition of sodium pyruvate, ferrous sulfate, or 3'3'-thiodiproionate supplements to MTSAYE did not show increased recovery (P>0.05). On each treatment and LMF combination tested, there was no significant difference between the log reduction of S. enterica and E. faecium NRRL B2354, indicating its suitability as a surrogate under the test conditions. / Master of Science in Life Sciences / Spices, dried herbs, and seeds have become popular throughout the world for enhancing the flavor of food, but may also harbor harmful bacteria, including Salmonella enterica. It is US federal law under the Food Safety Modernization Act that these foods are safe to eat straight from processors since these foods are typically consumed raw. Novel treatment methods are being tested to kill harmful bacteria on these dried foods without adding water including chlorine dioxide fumigation and hydrogen peroxide fumigation. However, these processes can injure the bacteria without killing them. These injured bacteria might not be counted using traditional means which could lead to overestimating the effectiveness of a treatment. Different media types, used as part of the process to count the number of bacteria in a sample, were tested to determine their effect on recovery of injured S. enterica cells. Furthermore, the bacterium Enterococcus faecium NRRL B2354 was tested against S. enterica to evaluate, if the former, a relatively harmless microorganism, could be used by food processing plants to determine that their treatment processes meets regulatory standards. More injured S. enterica cells were recovered from each non-selective media tested, compared to the selective media. Although there isn't a significant difference in injured S. enterica recovery between any supplemented non-selective media, any non-selective media recovers more sub-lethally injured cells, and would give more accurate bacterial counts. Results also indicated that E. faecium NRRL B2354 is a suitable surrogate to the pathogen S. enterica for spices and herbs processed under the same conditions.

Saturated steam (SS) and propylene oxide (PPO) fumigation are two common methods to improve microbiological quality and safety of tree nuts. Validation of these processes is needed to ensure adequate control of bacterial pathogens. Since pathogens cannot be studied in food processing environments, surrogates with resistance comparable to the pathogens needed to be identified. The objective was to investigate the suitability of Enterococcus faecium, Pediococcus acidilactici, and Staphylococcus carnosus as surrogate bacteria for Salmonella spp. on whole cashews and macadamia nuts, processed with SS or PPO. Whole cashews and macadamia nuts were co-inoculated with a cocktail of Salmonella enterica and one of the three potential surrogates. Nuts were dried to original aw, packaged in poly-woven bags (2.3 kg) and commercially processed using vacuum assisted steam at 80 ͦ C or PPO fumigation. Salmonella and the potential surrogates were enumerated by serial dilution, and plated onto TSA with overlay of XLT-4 (Salmonella) or media selective for the potential surrogates. Mean log reductions (CFU/g) of Salmonella and each potential surrogate were compared using a paired T-test. SS results: reduction of Salmonella (6.0 ± 0.14) was significantly larger than E. faecium (4.3± 0.12), or P. acidilactici (3.7± 0.14) on whole cashews. Salmonella (5.9 ± 0.18) was significantly larger than P. acidilactici (4.4± 0.18) on whole macadamia nuts. PPO results: reduction of Salmonella (7.3 ± 0.19) was significantly greater than E. faecium (6.4± 0.31), or P. acidilactici (6.3± 0.33) on whole macadamia nuts. Reduction of Salmonella was significantly greater than E. faecium and P. acidilactici reduction on cashews. P. acidilactici may be considered a surrogate for Salmonella reduction on whole macadamia nuts and whole cashews processed using SS at 80 ͦ C. E. faecium and P. acidilactici may be considered surrogates for Salmonella reduction on whole macadamia nuts and whole cashews processed using PPO. Reduction of St. carnosus exceeded that of Salmonella indicating it is not a suitable surrogate for Salmonella using either processing intervention. / Master of Science in Life Sciences / Tree nuts are produced and consumed worldwide, playing a role as a snack or ingredient in foods in several cultures. Traditionally, tree nuts have been believed to be microbiologically safe due to their composition and lack of water available to harbor pathogenic bacterial growth. However, recent years have proven to be problematic for many tree nuts and nut products, numerous recalls have occurred in the United States for verified contamination of <i>Salmonella</i>. Since <i>Salmonella</i> can be found in food systems worldwide, and is a leading cause of foodborne illness due to bacterial contamination of food, steps must be taken to improve the safety of nuts grown locally and imported products. With several processing options for tree nuts, two that are commonly practiced to reduce microorganisms include a fumigation of product using propylene oxide (PPO) and a thermal inactivation treatment through use of saturated steam. The comparison of <i>Salmonella</i> inactivation and non-pathogenic surrogate bacteria on cashews and macadamia nuts, being processed in these two manners, was investigated. Possible bacteria strains that were investigated for surrogacy were <i>Enterococcus faecium</i> NRRL B2354 (ATCC #8459), <i>Pediococcus acidilactici</i> (ATCC #8042), and <i>Staphylococcus carnosus</i> (ATCC #51365). Surrogates are bacteria that have similar inactivation characteristics to <i>Salmonella</i> when processed, that can be purposely introduced before processing to ensure inactivation of <i>Salmonella</i> and is harmless if consumption occurs. Studies continue to ensure safety of tree nuts as well as complying with pending and future regulations.

Orientador: Eleni Gomes / Banca: Mirna Helena Regali Seleghim / Banca: Eduardo Alves de Almeida / Resumo: Os basidiomicetos são conhecidos por sua capacidade em degradar a madeira. Essa característica deve-se à sua habilidade em oxidar a lignina presente nas plantas. Essa molécula é um composto recalcitrante formado por sub-unidades de fenilpropanóides e as enzimas capazes de degradá-la são conhecidas conjuntamente como enzimas ligninolíticas. Tais enzimas são inespecíficas. Essa característica confere-lhes o seu emprego na oxidação de compostos que possuem estrutura molecular semelhante à da lignina, tais como pesticidas e hidrocarbonetos poli-aromáticos, que possuem anéis aromáticos em sua estrutura. Dessa forma, é possível utilizar tais enzimas na degradação desses xenobióticos. Alguns basidiomicetos possuem a habilidade de crescer em meio com baixa atividade de água. Assim, é possível usá-los em processos de biorremediação de ambientes que possuem uma alta concentração de solutos, como em meios com alta salinidade. Esse trabalho teve como objetivo o estudo da capacidade de crescimento em meio líquido agitado com baixa atividade de água do basidiomiceto Dacryopinax elegans SXS323, promovida pela adição de cloreto de sódio 0,5 M, manitol 0,5 M ou glicerol 0,5 M ao meio de cultura; a produção de enzimas ligninolíticas e o seu poder de degradação do herbicida diurom nessas condições. Verificou-se que o basidiomiceto foi capaz de crescer sob todos os meios testados, sendo este mais pronunciado quando manitol ou glicerol foram acrescentados ao meio, averiguando-se um estímulo de crescimento. O manitol e o glicerol serviram como fonte secundária de carbono. O cloreto de sódio inibiu seu crescimento. Outro fator de inibição do crescimento foi a adição de diurom. A produção de enzimas ligninolíticas foi muito baixa, com pico de atividade de 3,67 U . L -1 com 144 horas de cultivo em meio com glicerol 0,5M. Apesar disso, detectou-se degradação... (Resumo comp'leto, clicar acesso eletrônico abaixo) / Abstract: The basidiomycetes are known for their ability to degrade wood. This feature is due to its ability to oxidize lignin of plants. This molecule is a compound formed by recalcitrant sub-units of phenylpropanoids and enzymes able to degrade it and are known as ligninolytic enzymes. These enzymes are nonspecific. This characteristic provides them the capability of oxidation of compounds that have molecular structures similar to lignin, such as pesticides and polyaromatic hydrocarbons, which possess aromatic rings in their structure. Thus, it is possible to use such enzymes in the degradation of xenobiotics. Some basidiomycetes have their ability to grow in media with low water activity. Thus, it can be used in bioremediation process in environments that have a high concentration of solutes, such as hypersaline environments. This work aimed to study the capability of the basidiomycete Dacryopinax elegans SXS323 to grow in liquid shaken with low water activity, promoted by the addition of sodium chloride 0.5 M, mannitol 0.5 M or glycerol 0.5 M to the medium culture, the production of ligninolytic enzymes and their degradation capability of the herbicide diuron in these conditions. It was found that the basidiomycete was able to grow in all media tested. The growth was most pronounced when mannitol or glycerol was added to the medium, probably working as a stimulus for growth, and were used as a secondary source of carbon. Sodium chloride inhibited its growth. Another factor inhibiting the growth was the addition of diuron. The ligninolytic enzyme production was very low, with peak activity of 3.67 U. L -1 with 144 hours in medium with glycerol 0.5 M. Nevertheless, we detected the degradation of the herbicide, registering 32.63% (glycerol 0.5 M, 192 hours), 25.47% (mannitol 0.5 M, 192 hours) 20.51% of reduction in medium with glucose 5.0 g . L -1 (240 hours)... (Complete abstract click electronic access below) / Mestre

Low water activity foods (LWAF), specifically nuts and dried fruits, have been generally considered safe because they do not support the growth of foodborne pathogens. However, many pathogens have been noted to survive in LWAF for considerable periods of time, and a number of recent outbreaks and recalls have implicated various types of nuts and dried fruits. The Food Safety Modernization Act requires food processors to develop preventive control plans that make ready-to-eat LWAF safer for consumers. The presented research was designed to investigate several aspects of LWAF safety by evaluating a steam process as a strategy to remove pathogen contamination from LWAF, modeling the inactivation of such treatments, and studying the thermal resistances of two E. coli strains in low-water activity solutions. Low-temperature, vacuum-assisted steam (vacuum-steam) was evaluated as a potential intervention and preventive control to remove pathogens from the surface of LWAF without using high-heat treatments that could damage product quality. The presented work examined the efficacy of vacuum-steam (<85°C) as a means to decontaminate the surface of whole macadamia nuts, dried apricot halves, and raisins from Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) contamination. The low-temperature steam treatments successfully reduced all pathogens by >4 log CFU/g from the surfaces of the foods. Additionally, Pediococcus acidilactici, proved to be a surrogate organism for these pathogens and could be used to challenge and validate similar treatments within processing plants. The data were fit to models, which showed that food type significantly impacted the fit, with the Weibull model best describing bacterial inactivation kinetics on raisins and macadamia nuts, and the Gompertz model best describing reductions on the apricot halves. The models were challenged for validation of their abilities to predict times required for 3-log reductions using internal and external datasets, determining the usefulness to industry members who wish to design similar thermal treatments for LWAF. Comparing predicted values from internally constructed models to observed values generated from external data, models were shown to be limited in scope and application and could only be applied to pathogen inactivation on different LWAF or thermal processes in certain circumstances. First-order and Weibull model predictions of bacterial reductions on dried apricots had varied success in predicting times for 3-log reductions on other thermally treated LWAF. However, the models of bacterial reductions on thermally treated macadamia nuts frequently overestimated the times required for 3-log bacterial reductions for other LWAF. In an effort to understand the effect that reduced water activity has specifically on STEC, two strains were investigated for induced thermal resistance due to osmotic stress. Thermal resistance of STEC strains (O121:H19 and O157:H7) were evaluated on the basis of strain variation, culture preparation, and water activity (D- and z-values). At the lowest treatment temperature (56°C), O121 displayed greater heat resistance than O157, and the broth-grown samples exhibited greater heat resistance than the lawn-grown cells, but significant differences were not observed at higher temperatures. Samples in reduced-water activity solutions displayed reduced thermal resistance at 56°C, but the z-values were 29-43% higher than those of high-water activity samples. While water activity has been shown to impact thermal resistance of pathogens, comparisons of STEC thermal resistance according to the D- and z-values revealed that other factors also play roles in pathogen thermal resistance on LWAF. Results from the collection of experiments conclude that efficacy of thermal treatments is impacted by the physiological state of the cells, stress experienced in the food matrix, and characteristics of the food, including water activity and composition. / Doctor of Philosophy / Consumers expect foods they purchase to be safe to consume by themselves and family members, particularly those that are ready-to-eat with no additional cooking requirements. Many of these foods are low-water activity foods (LWAF), like nuts and dried fruits, with very little water content that could be used by bacteria. These foods may be preferred snack foods due to their affordability, long shelf lives, and health benefits over other types of snack foods. Until recently, LWAF were generally considered safe because they do not support the growth of foodborne pathogens due to the lack of moisture or water within the food. However, a number of recent outbreaks related to various types of nuts and dried fruits have proven that many pathogens can survive in dried foods, even if not actively growing, for considerable amounts of time. Designed to address these types of food safety issues, the Food Safety Modernization Act recognizes risks associated with foods and responded with regulations requiring food processors to take steps to make ready-to-eat LWAF, like nuts and dried fruits, safer for consumers. A popular strategy is to treat foods with heat to destroy pathogens, however the quality attributes of some nuts and dried fruits could be damaged by high-heat treatments like roasting. An alternative process uses a vacuum to form steam at lower temperatures, allowing for efficient heat transfer through water droplets to the surface of the foods, thus causing less damage to the foods without introducing too much moisture. This research evaluated how this process could be used by food processors to remove harmful bacteria from the surfaces of whole macadamia nuts, dried apricot halves, and raisins. Results indicated that the low-temperature steam treatments successfully reduced Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) by >4 log CFU/g (>99.99%) from the surfaces of the foods. Additionally, a nonpathogenic lactic acid bacterium, Pediococcus acidilactici, exhibited similar or greater heat tolerance, which would allow food processors to use it as a substitute, or surrogate, for in-plant studies without introducing harmful bacteria into the food processing environment. Mathematical models were used to describe the trends of bacterial death due to the steam treatments, and the results indicated that the type of food significantly impacted the reduction of bacteria. The models were tested using additional data collected within our own laboratory, as well as others. Results indicated that some of the models could be used as predictors of bacterial death for similar LWAF but can only be applied with caution and consideration for the type of food and process. Additionally, two different E. coli strains associated with outbreaks (O121:H19 and O157:H7) were investigated to understand impacts of strain variation, growth method, and water activity on thermal resistance. Some differences in heat resistance were observed between the strains and between the growth methods. Additionally, the reduced water activity seemed to decrease the bacteria's ability to withstand some heat treatments. Overall, thermal resistance studies indicated that several factors, in addition to water activity, impact pathogens' development of resistance to heat treatments. The experiments' results show that there are complex relationships between bacteria and the food they inhabit. Food processors must consider these relationships in order to design the best thermal processes to make LWAF safe for consumers.