Chitin and chitosan are linear polysaccharides consisting of acetyl-glucosamine and glucosamine units with many potential applications in biomedicine, agriculture and food industry. In the food industry, chitosan can be used as antimicrobial agent and to create active packaging to improve food quality and extend shelf life. Commercial applications of chitosan are closely associated with its functional properties and biological activity, which are primarily governed by two structural properties: degree of acetylation (DA) and molecular weight (MW). The overall goal of this study was to develop methods for tailored modification of DA, MW and functionalities of chitosan. The research was conducted in four phases. In the first phase, an accurate and rapid method to determine DA for both chitin and chitosan was developed. By employing concentrated phosphoric acid as a solvent for highly acetylated samples and by optimizing the analytical parameters through investigation of underlying chemical reactions, it was the first time that the DA of both chitin and chitosan could be analyzed by a single method in an accurate, rapid and economical way. In the second phase, high intensity ultrasound (HIU) was investigated as a pretreatment method for the deacetylation process of chitin, and data indicated that HIU pretreatment of chitin flakes lasting up to 30 minutes was insufficient to change the DA of the resulting chitosan. Since the HIU treatment has been considered as an efficient way to modify the MW of polysaccharides in general, the third phase of this study focused on tailored modification of chitosan MW with HIU process. The factors that influence the ultrasonic degradation process of chitosan were investigated and the results showed that the MW, radius of gyration and polydispersity of chitosan were efficiently reduced, whereas the chain conformation and DA were unchanged after sonication. The degradation of chitosan by ultrasound was primarily driven by mechanical forces and degradation mechanism could be described by a random scission model. The degradation process was affected by ultrasound intensity, solution temperature, polymer concentration and ionic strength, while acid concentration had little effect. Additionally, the data indicated that the degradation rate coefficient was affected by the degree of deacetylation of chitosan and independent of the initial molecular weight. In the fourth phase, hydrophobic modification of chitosan (HM-chitosan) was performed to probe new way to improve the functionality of chitosan. The physicochemical properties and antimicrobial activity of HM-chitosan and HM-chitosan based films were investigated, and the data indicated that hydrophobic modification introduced thermal responsive and self assembly properties, while maintained the antimicrobial activity of chitosan. Furthermore, the water vapor permeability and mechanical properties of HM-chitosan based films decreased. Contributions of this research include developing a practical method for the DA analysis, elucidating the effects of HIU pretreatment on the deacetylation process and factors that affect the ultrasonic degradation process of chitosan, along with the effects of hydrophobic modification on the functionalities of chitosan and chitosan films.

This purpose of this investigation was to use DNA microarrays to study gene expression in E. coli O157:H7 under refrigerated and acidic conditions. Total RNA from E. coli O157:H7 grown to 7 log CFU/mL under control (37 °C, pH 7), refrigerated (10°C, pH 7), acid adapted (37°C, pH 5.5, then lowered to 3.5), and acid shocked (37°C, pH 7, then lowered to 3.5) conditions was extracted using an optimized Qiagen RNeasy procedure. Total RNA was converted to cDNA, labeled, and hybridized onto an Affymetrix GeneChip® E. coli Genome 2.0 Array. Results were analyzed using Analysis of Variance (ANOVA) and a t-test with significance set at p<0.01 and an expression cut-off of 2-fold difference in gene expression. Expression of selected genes, including an internal control (hemX) was confirmed using real time reverse transcriptase PCR. Microarray results revealed 293 down-regulated and 375 up-regulated genes. Cold shock genes cspE, cspA, cspG, and cspH were down-regulated; recA and SOS DNA repair genes uvrB, yebG, ruvA and B, lexA, and dinl were up-regulated. Expression of fhuA (outer membrane protein) and napG (electron transport) genes was 115- and 70-fold greater under refrigeration. Microarray analysis revealed that genes involved in DNA binding, biosynthesis, iron, transport and cellular metabolism were significantly up-or down-regulated in acid adapted cells. Genes related to iron uptake and transport (cyo, ent, fhu, fep, and feo operons) were up-regulated under acid shocked conditions. For acid adapted conditions, expression of cfa (cyclopropane fatty acid biosynthesis; decreases membrane fluidity and increases acid resistance) and genes involved in the glutamate decarboxylase acid resistant systems were significantly up-regulated. The virulence genes eae, stx1, and stx2 were not differentially expressed under either condition.

Food quality is often times measured by the way one perceives the food, particularly with respect to color and texture. Color quality often pre-determines expectation making it an important parameter to understand. The same can be said for textural properties of food. Maintaining color and texture are just two ways in which the shelf-life of food can be measured. One particular product that has experienced problems in this area is processed cheese spread in which the US military uses as part of the Meal, Ready-to-Eat (MRE) rations. The cheese spread is one of the most highly accepted products in the MRE’s; therefore, research was necessary to determine formulary changes that could be made in order to improve product quality and increase the parameters of its shelf life. Studies were done to determine the cheese-age effect and ingredient effects for the addition of vitamins, colorants, emulsifiers, and stabilizers. The greatest improvement for the problems of hardening and darkening over time was observed when vitamins were removed from the product. Colorants were studied in the cheese spread, as well as in model systems. Carotenoid pigments were selected to determine stability against the effects of light and oxygen, and to measure antioxidant capacity after exposure to ozone. These compounds are responsible for the yellow, orange, and reds observed in fruits, vegetables, and some algal species. Extraction from the natural source has made carotenoid pigments commercially available to the food industry. Environmental influences such as atmosphere and lighting do affect the stability of carotenogenic compounds by causing structural degradation which in turn causes changes in antioxidant abilities.

This investigation was conducted to address the issue of musty bean products. The main objectives of this study were to: (1) identify the source of the musty, off-flavor in processed navy beans, (2) determine and identify the associated off-flavor compounds (geosmin and 2-methylisoborneol (2-MIB)), and (3) isolate the microorganism(s) responsible for production of the compounds. To confirm the presence of geosmin and 2-MIB in processed baked beans, an automated gas chromatographic method employing solid phase micro-extraction (SPME-GC-MS) was used. Artificially musty baked beans, known musty beans, and raw ingredients (bacon, navy beans, and water) were analyzed. Results of this investigation revealed that navy beans were the source of the musty compounds. 2-MIB and geosmin were detected in artificially musty baked beans, and 2-MIB also was detected in control beans (non-musty). Neither geosmin nor 2-MIB was detected in smoked bacon or water. Results of the analysis identified geosmin and 2-MIB as the musty-odor compounds associated with processed navy beans, with moldy navy beans being the source. Aspergillus flavus, Penicillium commune, and Penicillium expansum were isolated from moldy navy beans. An additional experiment was performed to determine the production of geosmin and/or 2-MIB by the isolated molds. Non-moldy navy beans were inoculated with spore suspensions from A. flavus, P. commune, or P. expansum, incubated for 10 days at 25°C, and samples were analyzed for mold counts, geosmin, and 2-MIB.

The use of modeling techniques for safety and risk prediction in the food supply is a common practice. Factors affecting microbial heat resistance include those inherent to the organism, environmental conditions and the intrinsic properties of the heating menstruum. Varying physiological states of microorganisms could affect the measured response and add uncertainty to results from predictive models. Inactivation tests were performed using Escherichia coli strain K12 and E. coli O157:H7 for various growth conditions: traditionally or statically grown cells, chemostat‐grown cells, and chemostatgrown cells with buffered feed media. Heating menstruum was non‐buffered 0.1% peptone, 0.1 M phosphate buffer (pH 7.0), a simulated beef broth (pH 5.9) and actual beef broth obtained from 93% lean ground beef. Thermal inactivation of the cells was carried out at 58, 59, 60, 61 and 62°C and recovery was on a non‐selective tryptic soy agar. Chemostat cells were significantly less heat resistant than the traditional or buffered chemostat cells at 58°C. Shape response was also significantly different, with traditionally‐grown cells exhibiting reducing thermal resistance over time and chemostat cells showing the opposite effect. Buffering the heating menstruum to ca. pH 7 for both traditionally‐grown and chemostat cells resulted in inactivation curves which showed less variability or scatter of data points. Non log‐linear regression analysis resulted in the most accurate fit in most cases. There were significant differences in thermal resistance when cells were thermally treated in either simulated or actual beef broth mixtures compared to laboratory diluent.

Enterobacter sakazakii is considered an emerging opportunistic pathogen associated with sporadic life-threatening bacterial infections in neonates linked to the compsumption of contaminated infant formula [Stoll et al., 2004]. In 2001 a neonate fatal infection associated with the presence of E. sakazakii in infant formula occurred in the neonatal intensive care unit (NICU) of the University of Tennessee Hospital [Himelright et al., 2002], as a result of this outbreak, the hospital made several policy changes and requested the Food Safety Center of Excellence of University of Tennessee to analyze the growth pattern of this microorganism at the conditions maintained in the hospital. The objective of this study was to analize E. sakazakii growth profile during preparation and administration of formula, as well as E. sakazakii tolerance to chlorine sanitizers widely used in hospital settings.Our results showed that if the starting temperature of the formula at the time of administration was 6 oC, the formula reached 25 oC in a period of four hours. Once contaminated formula reach 25 oC the generation times can decrease to less than one hour. We also noted that cells organized in colonies or in contact with solid surfaces had a higher resistance to chlorine sanitizers than those of planktonic cells, this phenomena could be explained by the expression of genes triggered by the physical contact between cell and surface.

Chitosan films prepared with addition of other polymers have been widely studied for their modified properties. In this dissertation, poly (ethylene oxide) (PEO) and poly (N-vinyl-2- pyrrolidone) (PVP) were blended with chitosan. The objectives of the study were (1) to investigate the the effects of film thickness, blend ratios, and preparation methods on the physical, and mechanical properties and functional performance of chitosan/PEO films, and (2) to compare characteristics and functional properties of chitosan/PVP and chitosan/PEO films. The results demonstrated that regular cast chitosan/PEO films have altered properties than films produced from either polymer alone. Regardless of molecular weight, chitosan decreased the tendency to spherulitic crystallization of PEO. Production of ultra-thin chitosan and chitosan/PEO films with thickness below 80 nm was possible by spin-coating on silicon wafers. The increase of PEO content did not affect thickness of the films but the surface of corresponding films became rougher probably due to formation of PEO crystallites. Comparing the functional properties of thick, thin and ultra-thin chitosan/PEO films, the latter showed a significantly higher chromium binding capacity compared to the regular cast films. However, ultra-thin chitosan/PEO films did not show significant antibacterial properties due to their extremely low weight. A decreased film-forming time, especially in the spin-coating method, greatly reduced extent of film crystallization. Incorporation of PVP or PEO into chitosan films reduced the yellowish color and made films easier to puncture and tear. Although chitosan/PEO blend films showed lower water vapor permeability (WVP) values than chitosan/PVP films, blending chitosan with hydrophilic polymers was not an effective way to significantly improve the WVP. Replacing even 50% of chitosan with PVP or PEO in chitosan films did not significantly decrease the metal-binding and antibacterial properties of the films. Since synthetic polymers are less expensive than biopolymer chitosan, blending chitosan and synthetic polymers could reduce the amount of chitosan and lower the production cost with no effect on functionality of the films. Chitosan/PVP and chitosan/PEO blend films have the potential to be used in the food industry as active packaging materials to inhibit food borne pathogens and as absorbent to bind heavy metal from the environment.

This dissertation reports on the use of the casein micelle as an encapsulation and potential delivery system for a low molecular weight hydrophobic compound, triclosan. The ability of the casein micelle to serve in this capacity was tested in a series of experiments investigating: 1) micelle dissociation to expose hydrophobic binding sites of interaction, 2) encapsulation of triclosan, 3) the release of triclosan under various conditions, and 4) the intercellular delivery of triclosan to human liver cells in vitro for the inactivation of a malaria-causing parasite. Casein micelle dissociation and reassociaiton was achieved through high pressure homogenization and solvent-mediated pressure-induced dissociation. Triclosan was found to naturally associate to casein micelles in skim milk (up to 70%) and this association is enhanced by 30% at homogenization pressures reaching 300 MPa. The release of triclosan is governed by the disruption of micelle integrity, i.e., the enhancement casein-solvent interactions, where triclosan likely exists as a triclosan-protein complex as opposed to free triclosan. In the presence of milk, the release of triclosan is prevented in simulated gastric solutions but released in a simulated intestinal solution, demonstrating the ability of to milk protect triclosan from the acidic environment of the stomach and promote its release in the intestine, a measure of bioaccessibility. The intercellular delivery of triclosan was most affected by the type of milk protein. Triclosan transported within β-casein isolates where most sufficient in preventing the growth and development of Plasmodium berghei, the rodent malaria parasite, in human liver cells. Triclosan carried within milk digestates were also efficient in delaying parasite growth, whereas triclosan carried within milk serum by serum proteins where completely ineffective. Triclosan in the presence of milk also provided a protective effect on host cells from the acute toxicity of triclosan in free form. These experiments introduced new functional properties of the casein micelle. The casein micelle has the ability to serve as an encapsulation system and casein proteins, as a potential delivery system. This introduces new functional properties of the casein micelle for use novel usage within the food, pharmaceutical, and/or cosmetic industry.

Decreased risks of chronic illnesses, such as cancer, occur with increased consumption of dietary antioxidants. Vegetables are a particularly rich source of dietary antioxidants but these are primarily water soluble compounds. This research determined effects of microwaving or boiling on the antioxidant capacities of commercially frozen vegetables. Hydrophilic components were extracted by Acetone/Deionized water/Acetic Acid (700:295:50, v/v) from commercially frozen broccoli, carrots, sweet corn, and sweet peas before and after microwaving for 5 min or boiling for 10 min. The Oxygen Radical Absorbance Capacity (ORAC) assay was employed to determine the antioxidant capacity. Additionally, color and texture analyses were performed. ORAC values from uncooked, microwaved or boiled broccoli were 11.33, 8.04 and 5.72 μmol TE/g; ORAC values for peas were 10.2, 5.14 and 2.43 μmol TE/g; ORAC values for corn were 6.32, 8.12 and 4.45 μmol TE/g; and ORAC values for carrots were 2.95, 4.00, and 2.39 μmol TE/g. No significant ORAC and texture correlations were determined. The only significant color and ORAC correlation was for the a* value of peas (p<0.05). A negative moderate correlation existed; therefore, greener peas had greater ORAC values. These results demonstrate that boiling vegetables for 10 min results in lower ORAC values and boiled broccoli, peas and corn contained significantly lower values (p<0.05) than uncooked broccoli or peas and microwaved corn. Boiled vegetables have been found to contain significantly lower water soluble nutrients due to loss into the cooking water. ORAC analysis of cooking water from each of the four vegetables verified the loss of antioxidant constituents since the water was found to contain increased antioxidant capacity. The greatest ORAC values were found in the water after boiling broccoli and the lowest ORAC values were found in the water after cooking carrots. Addition of antioxidant capacities of cooked vegetables in nutritional databases would be useful to consumers wanting to increase consumption of antioxidants.

Components of plant essential oil (PEO) extracts are known to have antimicrobial properties. However, their antimicrobial efficacy in food systems is low due to their hydrophobic nature and association with other food components. Incorporation of PEO components into an appropriate carrier may offer a potential solution to improve their activity in food systems. This study was conducted to determine the effect of PEO components (thymol, eugenol, linalool, carvacrol, and cinnamaldehyde) incorporated into zein coating on inactivation of Listeria monocytogenes on a ready-to-eat meat model, frankfurters (hot dogs). Hot dogs were inoculated with 7 log CFU/sample and dipped into prepared PEO-zein coatings. Samples were vacuum packaged and stored at 10ºC for 9 days or 4ºC for 4 weeks. Survival of L. monocytogenes was investigated by direct plating onto modified Oxford agar. Enrichment using UVM broth and Fraser broth was conducted when the pathogen was not detected by direct plating. Generally, results show that the PEO loaded coatings are effective against L. monocytogenes at 10 and 4ºC. When compared to the coating control (zein coating without PEO) at 10ºC, coatings loaded with 10% cinnamaldehyde and 1% carvacrol showed the greatest inhibitory effect and suppressed growth of L. monocytogenes by 2.4 and 2.1 log CFU/sample, respectively, after 9 days of storage. At 4ºC, the coating loaded with carvacrol was most effective at suppressing growth of L. monocytogenes (1.54-log reduction). No or little dose-response association between PEO concentration and antimicrobial activity was observed in the study. While further research is still required, this study indicates that incorporation of PEOs in corn zein to be used as an edible coating has a high potential to enhance the safety in ready-to-eat meat.