<p>The attached document contains the dissertation of Clare Jensen, including descriptions of all procedures used in the associated research studies.</p>

<p dir="ltr">The impacts of processing on protein structure are of broad interest to the food science community including ingredient producers, product developers, and researchers. Processing and isolation steps induce protein structural changes which occur due to temperature based, shear, and chemical inputs, leading to denatured protein with different functionalities. However, exploration of the protein folding landscape as a way to intentionally modify protein conformation is not widely understood in food science. This particularly applies to cold denaturation, which is the structural changes in protein as the result of low temperature treatments.</p><p dir="ltr">This work has two primary goals. The first was to develop understanding of protein conformations resulting from cold denaturation and its implications for food textural properties. Pea protein was selected for this work since it is a source of plant-based protein that has recently grown in popularity and contains many hydrophobic amino acids that would make is susceptible to cold denaturation. Cold denaturation was studied using physicochemical techniques including differential scanning calorimetry, Fourier transform infrared spectroscopy, zeta potential, fluorescence spectroscopy, dynamic light scattering, and rheology. These techniques are used to characterize untreated pea protein, and proteins that have been modified using different combinations of ethanol, shear forces, acidic conditions, extrusion, and temperatures below 0°C. Significant physicochemical differences are found as the result of low temperatures, driven by an increase in surface hydrophobicity and electrostatic interactions. These differences led to protein gelation through hydrophobic forces, changing the nature of gels. Similarly, the increase in protein hydrophobicity leads to more stable emulsions from these products and unique fatty extrudates.</p><p dir="ltr">A second aim of this work developed bioinformatic models to interpret physiochemical data and provide mechanistic understanding of the process, as well as predict functional properties based on protein models. Strong correlations are found for the zeta potential, secondary structure, hydrogen bonds, and surface hydrophobicity. These models are used to convert data into physicochemical energy and used to provide reasonable estimates of mechanical properties of pea protein in extrusion, gelation, and emulsification. Together, this work shows that cold denaturation may be a useful tool for food product developers creating fatty and creamy textures. It also suggests bioinformatic modeling as a tool to estimate protein functionality, which could lead to tremendous time savings in process and product design.</p>

<p dir="ltr">Global food demand increases rapidly as a result of continuously growing population has raised severe concerns with food security. To overcome this critical challenge, food systems must be transformed to produce food with not only higher yield but also better nutritional quality. Therefore, food processing, as a critical step in food production chain that turn agricultural products into food, needs to be innovated through applications of cutting-edge technologies.</p><p dir="ltr">Microbubbles (MBs) are tiny gas-filled bubbles with distinctive physicochemical characteristics, including slow rising speed and long lifetime in liquid, large surface area per unit of gas volume, high internal pressure, high gas dissolution rate, hydrophobic and negatively charged surface and production of reactive oxygen species. Additionally, MB dispersion can enhance the heat and mass transfer properties of liquid. These features have led MBs to numerous applications in the fields of disease treatment, anaerobic digestion, and wastewater treatment, however, their applications in food processing have not thoroughly explored.</p><p dir="ltr">In this dissertation, MB technology was applied to different unit operations of food processing, including freezing, concentration and extraction, and the effects of MBs on process efficiency and food product quality were comprehensively studied. In the first study, MB-infused freezing medium was used for grape tomato immersion freezing. MBs markedly reduced the drip loss of tomato by 13.7–17.0% and improved its firmness, which were correlated to the accelerated nucleation process and formation of small ice crystals during freezing. The impact of MBs on water evaporation during apple juice concentration was investigated in the second study. MBs dramatically enhanced water evaporation, and concentration at bubble gas temperature of 40 °C and juice temperature of 70 °C showed the largest increase in the evaporation rate, by 104%. Moreover, although air-MBs showed an oxidation effect on both frozen tomato and concentrated juice, N₂-MBs were found to be an ideal alternative which much better preserved the nutritional values of processed foods. Lastly, MBs and cold plasma-MBs were incorporated into citric acid solution for extracting pectin from apple pomace. MBs present in extracting solvent increased the extraction yield by 18–21%, and extraction with plasma-MBs showed even higher yields by up to 30%. Additionally, MB and cold plasma-assisted extraction were found more effective in extracting complex RG-I pectin.</p><p dir="ltr">This dissertation develops various approaches to incorporating MBs into conventional unit operations and enhancing their performance. With these novel applications explored, MB technology will not only increase the productivity but also reduce the energy, water and chemical use of food processing. Ultimately, MB-assisted processes are expected to play an important role in improving the sustainability of the food industry.</p>

<p dir="ltr">Liquid hydrogen peroxide (LHP) and vapor hydrogen peroxide (VHP) efficacy as a sterilant for <i>Bacillus atrophaeus</i> and <i>Geobacillus stearothermophilus</i> spores in aseptic packaging systems under commercial sterilization conditions were evaluated in this research. The work centers on quantifying and modeling the kinetic parameters that impact peroxide sterilization efficacy, including the D and z values, that relate to the change in concentration required for a 1-log reduction in spore population and a novel Z_conc parameter This comprehensive study is divided into four key investigations, each contributing critical insights to the overall understanding of peroxide sterilization processes.</p><p dir="ltr">The first study examined the inactivation kinetics of <i>B. atrophaeus</i> spores in liquid hydrogen peroxide. By evaluating different concentrations (20%, 28%, and 33%) and temperatures (up to 82.2°C), the study revealed that higher concentrations and elevated temperatures significantly enhanced spore inactivation. The Weibull model provided a more accurate fit for the data, indicating a non-linear relationship between spore reduction and exposure time.</p><p dir="ltr">The second part of the research explored the use of VHP for sterilizing <i>B. atrophaeus</i> spores. With VHP concentrations of 2500 ppm and 4450 ppm at various temperatures, the study demonstrated that higher concentrations and temperatures are highly effective for spore inactivation. Both log-linear and Weibull models accurately described the inactivation kinetics, with the Weibull model showing a slightly better fit, emphasizing the potential of VHP in achieving commercial sterility.</p><p dir="ltr">The third investigation focused on developing predictive models for VHP concentration and its efficacy in spore inactivation. The study evaluated VHP concentrations ranging from 2.32 mg/L to 7.35 mg/L and their impact on spore inactivation rates. The Weibull model proved particularly effective in predicting the inactivation of <i>G. stearothermophilus</i> and <i>B. atrophaeus</i> spores, offering a robust tool for optimizing sterilization protocols in aseptic packaging.</p><p dir="ltr">The fourth and final study of the research investigated the influence of surface roughness on spore survival during VHP sterilization cycles on plastic packaging materials. Artificial roughness on high-density polyethylene (HDPE) coupons was created using sandpaper with different grits. <i>B. atrophaeus</i> spores were applied to both roughened and smooth HDPE surfaces. The study finds that rougher surfaces provide more shelter for spores, reducing sterilization efficacy. For example, surfaces roughened with P-36 grit showed a 2.75 log reduction in spore count, whereas smoother surfaces with P-220 grit achieved a 4.42 log reduction. Contact angle measurements indicated that increased roughness led to more hydrophilic surfaces, with water contact angles decreasing from 149.7° for the pristine sample to 105.4° for the P-36 sample. Scanning electron microscopy (SEM) images confirmed that spores were more likely to reside in the valleys of rough surfaces, highlighting the importance of surface characteristics in optimizing VHP sterilization protocols.</p><p dir="ltr">The findings of this dissertation underscore the significant impact of hydrogen peroxide concentration, application conditions, and packaging material surface properties on the efficacy of spore inactivation during sterilization. By providing a comprehensive understanding of these factors, the research contributes to the development of optimized aseptic sterilization protocols, enhancing the reliability and safety of aseptically packaged food and pharmaceutical products. This work will ensure compliance with regulatory standards and improve food safety in commercial manufacturing, laying a solid foundation for future research and practical applications in VHP sterilization technology.</p>

<p dir="ltr">This dissertation delves into three projects within the realms of image processing, computer vision, and machine/deep learning. The primary objective of the first project is the detection of heavy metal particle concentrations using microfluidic paper-based devices. The second project revolves around the analysis of crystals within microscopic images. The third project centers around zero-shot action recognition in video sequences, utilizing a multi-modality deep learning framework that is refined through prompt tuning to enhance its performance.</p>

<p dir="ltr">This thesis explores the complex relationship between <i>Listeria</i> <i>monocytogenes</i> and food processing environments (FPEs), focusing on the persistence mechanisms of this pathogen despite rigorous sanitation efforts. <i>L</i>. <i>monocytogenes</i> is a significant public health concern due to its association with high mortality rates in vulnerable populations. Recent studies, including the first chapter of this thesis, have highlighted the challenges in eradicating this pathogen from FPEs, suggesting that biofilms play a crucial role in its persistence. Despite various strategies and enhanced sanitation protocols, the eradication of <i>L</i>. <i>monocytogenes</i> remains elusive, underlining the need for a deeper understanding of its biofilm-associated resilience. Chapter two synthesizes findings from a systematic review and meta-analysis of studies examining the microbial communities on FPE surfaces through metagenomics, aiming to uncover patterns that might explain <i>Listeria</i>'s long-term survival. In chapter three, the thesis delves into the nuanced role of biofilm composition and microbial diversity as factors enabling the persistence of <i>Listeria monocytogenes</i> in food processing environments (FPEs), despite comprehensive sanitation efforts. This section builds on the understanding that <i>L. monocytogenes</i> does not inherently form robust biofilms but thrives within complex microbial communities present on FPE surfaces. The findings presented in this thesis contribute to a more profound comprehension of <i>L. monocytogenes</i>' survival strategies, proposing a shift in focus towards the microbial ecosystem's metabolic interplays for the development of targeted control measures. This approach not only opens new avenues for research but also suggests practical implications for enhancing food safety protocols by considering the broader microbial dynamics at play within biofilms on FPE surfaces.</p>

<p>Maize is one of the top sources of food starch. Industrial use of starch is mostly in its native form and used due to their functional and structural properties. Native starch properties and functionalities have been altered using chemical. An alternative for the development of native starch substituents with desirable starch properties is the use of mutagenesis techniques to increase genetic variation in maize kernels. With this approach, a highly diverse library of native starches with different properties are produced. Traditional analysis of the functional and structural properties requires generous amounts of material as well as a time-consuming and costly breeding process to obtain enough kernels. To address this difficulty, high-throughput techniques are proposed for studying starch properties and functions which includes a 1) single kernel sampling method for the isolation of milligrams of starch, and techniques for studying starch based on functional properties, 2) retrogradation and 3) shear resistance, using low-volume low-concentration starch pastes.</p><p>First, three mechanical approaches were evaluated for the collection of endosperm samples from individual kernels: razor blade, 1.5 mm drill bit, and trephine bur. Furthermore, two methods for the isolation of crude starch from endosperm samples (steeping method and combination of proteases and sonication) were compared. In this study, the mechanical approaches were evaluated using the recovery rate, throughput, and germination rate of sampled kernels. Moreover, yield determination, particle size distribution, and morphological evaluation using a light microscope were performed on crude starch isolated from the endosperm samples. The use of trephine bur to collect endosperm samples and isolation of crude starch using protease digestion and sonication showed the best combination for a high-throughput setting. </p><p>Second, a high-throughput technique using milligram sample for the screening of retrogradation-resistant starch was evaluated by comparing two spectrophotometric techniques: turbidity method and molecular rotor (MR). MRs are fluorescent probes with high sensitivity to the viscosity of their environment, polarity of the media, molecular crowding, and free volume. After excitation, MRs relax through rotational movement and reduces the emission of fluorescence. In this study, hydroxypropylated waxy corn starch (WCS) and hydroxypropylated normal corn starch (NCS) were used and their retrogradation kinetics was compared with retrogradation kinetics of native WCS and NCS. </p><p>It was found that the molecular rotor 9-(2-carboxy-2-cyaovinyl)-julolidine (CCVJ) was effective to sense changes during slow retrogradation of amylose-containing starch pastes. Development of elastic modulus of retrograded NCS pastes obtained from dynamical rheology showed high correlation with the development of fluorescence intensity of the CCVJ. Furthermore, rate of retrogradation using fluorescence intensity was affected by the introduction of a retrogradation inhibitor, hydroxypropyl groups. Accelerated retrogradation of low-concentration WCS pastes was measured using the turbidity method and fluorescence intensity of CCVJ in a microplate. Accelerated retrogradation was performed by subjecting the low-concentration WCS pastes to six freeze-thaw cycles of -20 ºC for 1 hour and 30 ºC for 1 hour. Overall, development of turbidity resulted in the more sensitive technique to detect rate of retrogradation of amylopectin-containing starch. </p><p>The last part of this research studied the use of CCVJ as a technique to identify shear-resistant starch in starch slurries using milligram sample. For this purpose, WCS was cross-linked with sodium trimetaphosphate (STMP) and phosphoryl chloride (POCl₃). Low-volume starch slurries having CCVJ were prepared ranging from 0.5% to 1% starch concentration in a 96-well PCR plates and subjected to heat and shear treatments. It was found that fluorescence intensity measured in native WCS pastes were the lowest. Furthermore, fluorescence intensity of the CCVJ in the gelatinized starch increased as the amount of cross-linker increased in the cross-linked WCS. After shear treatments, the same trend in fluorescence intensity increase was recorded in all the crosslinked WCS. Results obtained using fluorescence intensity were compared with rapid viscosity analyzer (RVA) and images from microscope. Results obtained from both techniques corroborated the findings using fluorescence intensity.</p><p>In general, the findings of this research provide new insights into the possibilities of developing a high-throughput screening platform of milligram starch sample based on their physical properties. </p>

With the advent of industrialization and food processing techniques the sizes of the cereal bran have been drastically reduced. In my thesis, I have tested the effect, if any, of wheat bran and maize bran particle size, in vitro, on the gut microbiota community structure by 16S rRNA sequencing and their function, by Short chain fatty acids (acetate, propionate, butyrate) production. In turn, we also linked the microbiota and SCFA differences to different chemical composition amongst variously sized fractions of wheat and maize bran.

This thesis investigates a newly developed and patented technology for its ability to inactivate spore- forming bacteria and non-spore-forming microorganisms. This new technology “Pressure Assisted Thermal Sterilization©” (PATS) is based on the theory of the thermal expansion of liquids. The efficiency of inactivating spore-forming and non-spore-forming microorganisms by PATS was compared with the thermal treatment alone. A combination treatment consisting of high pressure processing and gaseous carbon dioxide was also investigated for its ability to inactivate bacterial spores in model and real food matrices. The structural damage caused by treatments to the spores and non-spore-forming bacteria was assessed by scanning electron microscopy. Geobacillus stearothermophilus spores suspended in Milli-Q water, UHT milk and pumpkin soup, treated by PATS were found to have significantly lower decimal reduction times (D values) compared with the thermal treatment alone. Spores suspended in UHT milk were more heat resistant compared with those in Milli-Q water and pumpkin soup. Bacillus cereus spores suspended in Milli-Q water and pumpkin soup treated with PATS were more effectively inactivated compared with spores treated by the thermal treatment alone. Clostridium botulinum spores in saline buffer subjected to PATS treatment were inactivated more effectively compared with the thermal treatment alone. Overall, the results show that PATS was a better processing technique for inactivation of bacterial spores compared with thermal treatment alone. However, PATS had no added benefit in inactivating the non-spore-forming bacteria Escherichia coli and Saccharomyces cerevisiae cells compared with the thermal treatment. A shelf life study showed that B. cereus spores in pumpkin soup retained a low spore count (<5 LogCFU/mL) for approximately 40 days in 30oC storage after treatment with PATS. No additional degradation of colour pigments of pumpkin soup and model pumpkin juice was observed following PATS compared with the thermal treatment. Spore-forming microorganisms can be resistant to pressure treatment alone, which limits the application of high pressure processing (HPP). Therefore, a combination approach was investigated. The mechanism of inactivating spores by combining HPP with other treatments is that the pressure assists in spore germination. Then a secondary treatment (thermal or CO2 gas) can be used to inactivate the germinated spores. A combined application of HPP and a consecutive CO2 treatment was investigated for the efficiency of spore inactivation. Results showed that HPP (200 MPa for 30 min) followed by a CO2 treatment inactivated Bacillus subtilis 168 in nutrient broth, tomato juice and liquid whole egg by 2.5, 1.0 and 1.5 LogCFU/mL respectively. These results indicated that this technique is inadequate for practical use. Scanning electron micrographs showed that pressure processing of B. subtilis 168 and B. subtilis natto spores resulted in deformation of the spore structure. This structural deformation of spores may have been due to water absorption during HPP and subsequent release upon decompression. PATS treated G. stearothermophilus and B. cereus spores were more severely damaged compared with the same spores which underwent thermal treatment alone. However, the extent to which E. coli and S. cerevisiae cells were damaged by both PATS and thermal treatment was similar.

This thesis investigates a newly developed and patented technology for its ability to inactivate spore- forming bacteria and non-spore-forming microorganisms. This new technology “Pressure Assisted Thermal Sterilization©” (PATS) is based on the theory of the thermal expansion of liquids. The efficiency of inactivating spore-forming and non-spore-forming microorganisms by PATS was compared with the thermal treatment alone. A combination treatment consisting of high pressure processing and gaseous carbon dioxide was also investigated for its ability to inactivate bacterial spores in model and real food matrices. The structural damage caused by treatments to the spores and non-spore-forming bacteria was assessed by scanning electron microscopy. Geobacillus stearothermophilus spores suspended in Milli-Q water, UHT milk and pumpkin soup, treated by PATS were found to have significantly lower decimal reduction times (D values) compared with the thermal treatment alone. Spores suspended in UHT milk were more heat resistant compared with those in Milli-Q water and pumpkin soup. Bacillus cereus spores suspended in Milli-Q water and pumpkin soup treated with PATS were more effectively inactivated compared with spores treated by the thermal treatment alone. Clostridium botulinum spores in saline buffer subjected to PATS treatment were inactivated more effectively compared with the thermal treatment alone. Overall, the results show that PATS was a better processing technique for inactivation of bacterial spores compared with thermal treatment alone. However, PATS had no added benefit in inactivating the non-spore-forming bacteria Escherichia coli and Saccharomyces cerevisiae cells compared with the thermal treatment. A shelf life study showed that B. cereus spores in pumpkin soup retained a low spore count (<5 LogCFU/mL) for approximately 40 days in 30oC storage after treatment with PATS. No additional degradation of colour pigments of pumpkin soup and model pumpkin juice was observed following PATS compared with the thermal treatment. Spore-forming microorganisms can be resistant to pressure treatment alone, which limits the application of high pressure processing (HPP). Therefore, a combination approach was investigated. The mechanism of inactivating spores by combining HPP with other treatments is that the pressure assists in spore germination. Then a secondary treatment (thermal or CO2 gas) can be used to inactivate the germinated spores. A combined application of HPP and a consecutive CO2 treatment was investigated for the efficiency of spore inactivation. Results showed that HPP (200 MPa for 30 min) followed by a CO2 treatment inactivated Bacillus subtilis 168 in nutrient broth, tomato juice and liquid whole egg by 2.5, 1.0 and 1.5 LogCFU/mL respectively. These results indicated that this technique is inadequate for practical use. Scanning electron micrographs showed that pressure processing of B. subtilis 168 and B. subtilis natto spores resulted in deformation of the spore structure. This structural deformation of spores may have been due to water absorption during HPP and subsequent release upon decompression. PATS treated G. stearothermophilus and B. cereus spores were more severely damaged compared with the same spores which underwent thermal treatment alone. However, the extent to which E. coli and S. cerevisiae cells were damaged by both PATS and thermal treatment was similar.