Validation of high protein chocolate chip cookies, crackers, and evaluation of thermal resistance characteristics of Salmonella, Shiga toxin producing Escherichia coli and Cronobacter sakazakii in bakery products and ingredients
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[EMBARGOED UNTIL 12/01/2026] Raw flour is a common ingredient in a variety of baked food products and may harbor foodborne pathogens, which can potentially survive and contaminate the final product if adequate thermal processing is not achieved during baking. The thesis covers chapters on validation of cookies formulated with milk protein concentrate (MPC80), whey protein concentrate (WPC80), and a standard flour-based control against Salmonella and Shiga toxin producing Escherichia coli (STEC). The fifth chapter is studying the baking validation and thermal inactivation parameter studies of Salmonella and STEC in cheddar cheese and whole wheat crackers. The sixth chapter is dedicated to study the survivability of Salmonella, STEC and Cronobacter sakazakii in whey protein concentrate during extended storage. For chocolate chip cookies baking, the maximum baking internal temperatures for cookies prepared with MPC80, WPC80, and the flour (as control) were 212.46± .25°F, 213.69±2.03°F, and 213.19±1.04°F, respectively. The humidity ratio for MPC80 chocolate chip cookies ranged between 0.0090±0.0005 and 0.0829±0.0044 kg moisture/kg dry air, whereas the humidity ratio for WPC80 and flour-based chocolate chip cookies ranged between 0.0110±0.0027 and 0.01045±0.0016, and 0.0103±0.0010 and 0.0952±0.0029, respectively. Water activity (aw) remained higher in both crust and crumb regions of MPC80 cookies compared to WPC80 and flour formulations. Proximate analysis showed elevated protein and moisture levels in MPC80 and WPC80 doughs relative to the control. Salmonella and STEC populations in all cookie doughs were initially greater than 6.8 log CFU/g and declined by more than 5 log CFU/g after baking and cooling. Thermal inactivation studies showed that the D-values for Salmonella at 55°C were highest in MPC80 cookie dough, followed by WPC80 and flour-based cookie dough. At 58°C and 61°C, the D-values for Salmonella were highest in WPC80 dough, followed by MPC80 and flour-based dough. The corresponding z-values were 8.75°C (MPC80), 8.72°C (WPC80), and 6.90°C (flour). For STEC, D-values were highest in WPC80 dough, followed by flour-based and MPC80 dough, respectively. The corresponding z-values were 8.36°C (WPC80), 7.42°C (flour), and 7.20°C (MPC80). The baking validation of cheddar cheese and whole wheat crackers against Salmonella and STEC evaluated the thermal and physicochemical properties of cheddar cheese and whole wheat crackers during baking, alongside the thermal inactivation kinetics of STEC and Salmonella. The baking profile showed maximum internal temperatures of 238.22°F (114.57°C) at 10.01 minutes for cheddar cheese crackers and 234.11°F (112.28°C) at 11.03 minutes for whole wheat crackers. Water activity (aw) decreased significantly throughout baking and cooling, with initial dough aw of 0.853 and 0.906 which dropped below 0.36 after cooling. The pH values declined slightly during cheddar cheese and whole wheat crackers baking, stabilizing post-cooling. The oven humidity ratio increased markedly during baking for both cracker types. The proximate analysis showed that cheddar cheese dough had a higher level of crude protein and fat, whereas whole wheat dough had higher fiber and moisture. Microbial analysis demonstrated substantial reductions in STEC and Salmonella populations in both cheddar cheese and whole wheat crackers, with final counts dropping below 1 log CFU/g after baking and cooling. Thermal inactivation assays yielded D-values ranging from approximately 2 to 14 minutes and z-values between 7.7 and 12.7°C, indicating variable heat resistance depending on the pathogen and dough recipe. The storage study of inoculated whey protein concentrate (WPC80) with Salmonella, STEC, and Cronobacter sakazakii for 120 days investigated the survival and thermal resistance of Salmonella, STEC, and C. sakazakii under ambient conditions. Inoculated WPC80 samples were analyzed at regular intervals for changes in bacterial populations, water activity (aw), pH, and thermal inactivation parameters (D- and z-values). All three pathogens exhibited significant reductions in viable cell counts over the storage period, with Salmonella, STEC, and C. sakazakii population decreased by 3.45, 2.99, and 3.67 log CFU/g, respectively. Water activity increased over time, likely due to repeated exposure to ambient humidity during sampling. A modest decline in pH was observed, particularly in STEC and C. sakazakii-inoculated samples. One-way ANOVA followed by Tukey's post hoc test (p ≤ 0.05) was employed to evaluate significant differences in survival, aw, pH, and thermal inactivation parameters. Thermal inactivation experiments were conducted using WPC80 powders inoculated with each pathogen and heated at 80, 85, and 90°C in circulating water baths. Linear regression was applied to determine D- and z-values. Thermal resistance varied among target pathogens with storage duration. Salmonella exhibited a progressive decline in both D- and z-values, indicating reduced heat resistance over time. In contrast, STEC maintained relatively stable thermal tolerance. The increase in C. sakazakii's z-values suggests an enhanced thermal tolerance during storage, which suggests the necessity for thorough risk assessment and optimizing product-specific baking parameters for safety.
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Ph. D.