Factors affecting lipid oxidation in oil-in-water emulsions were studied. Sodium dodecyl sulfate (SDS), polyoxyethylene (10 or 23) lauryl (Brij), and dodecyltrimethylammonium bromide (DTAB) were used to make anionic, non-ionic, and cationic emulsion droplets, respectively. Iron accelerated lipid oxidation in the emulsions with the oxidation rates of SDS $>$ Brij $>$ DTAB. Oxidation of SDS emulsion increased with decrease in pH in iron-added systems. Iron associated strongly with SDS-stabilized emulsion droplets but not with Brij and DTAB. EDTA ($\geq$ 1000 $\mu$M) decreased lipid oxidation to levels lower than no-added iron controls. This was due to the ability of the EDTA to remove both added and contaminating metals from the emulsion droplet surface as determined by changes in zeta potential. Gallic acid, methyl gallate, and gallamide were tested as anionic, nonionic, and cationic antioxidants, respectively. These galloyl derivatives exhibited both antioxidative free radical scavenging and prooxidative Fe$\sp{3+}$-reducing activity. Initial metal-reduction rate by the galloyl derivatives was higher at pH 3 than 7. Galloyl derivatives did not associate with SDS-stabilized emulsion but partition into Brij-stabilized emulsion droplets. Galloyl derivatives did not alter iron-droplet association by chelation. Charge status of the galloyl derivatives influenced their ability to partition into Brij-stabilized emulsion. Strong association of iron with emulsion droplets is responsible for the low oxidative stability of SDS-stabilized emulsions. The amount of iron commonly found as a contaminant in emulsion can significantly accelerate oxidation. The prooxidant activity of contaminating iron can be controlled by use of chelators and nonionic or cationic emulsifiers. The net antioxidant/prooxidant effect of galloyl derivatives in emulsions is a balance between their free radical scavenging and metal-reducing activity. This balance is influenced by factors including pH, emulsifier type, galloyl derivative concentration and physical location. This study shows that surface property of the emulsion droplets and interactions among emulsion droplets, antioxidants, prooxidants and other co-existing substances are critical to oxidative stability of food emulsions.

The dynamic behavior of water molecules was analyzed in multicomponent systems by $\sp1$H, $\sp2$H and $\sp{17}$O Nuclear Magnetic Resonance. Systems chosen were culture media for microorganisms. For the case of a fast exchange, evaluation of results obtained from $\sp{17}$O NMR suggested that the "anisotropic, two correlation time model" (Halle and Wennestrom, 1981; Belton et al., 1991) might be an appropriate model. The correlation time for the slow relaxing water component ($\rm\tau\sp{s}\sb{bw}$) was higher in NaCl solutions than in BHI of same concentrations, suggesting a stronger interaction of the water with NaCl than with the BHI solids. P$\sb{\rm bw},$ or the population of bound water (fast and slow) increased with increasing solid content. Water molecular mobility was studied in solid and semisolid systems (gums, with and without mannitol; cellulose, with and without sorbose) by applying solid state $\sp1$H and $\sp2$H NMR. No observable glass transition as analyzed by DSC and/or DMA was observed in the polymers used. However, at the molecular level, NMR mobility was observed consistently. The molecular mobility, as monitored by T$\sb2$ relaxation time, and liquid (or mobile) signal intensity increased with increasing moisture content. The mobile fraction increased greatly from 10% mobile at approximately 3% water, to close to 100% mobile at $\sim$12-16% moisture (T$\sb2$ relaxation time from $\sp2$H NMR was 100-700 $\mu$sec) in the xanthan and locust bean gum mixtures. In the case of cellulose, solid state $\sp1$H and high resolution $\sp2$H NMR data at 2-16% moisture content (dry basis) indicated not only anisotropic reorientation but also a slow exchange within the NMR time frame. In the presence of water-soluble solutes, water mobility was primarily affected by the amount of dissolved solute present. This is due to the osmotic competition for water; more water in a liquid state solubilizing the solute was found to play a major role with a significant increase in T$\sb2$ relaxation time. In a diluted solution, NMR T$\sb2$ relaxation time was dependent slightly on solid concentration. The NMR water molecular mobility results were correlated with microbial survival and growth studies (done by other researchers), as compared with other parameters, such as water activity, water content and kinematic viscosity. In a fast exchange (liquid systems, BHI and NaCl), NMR mobility was less influential and a$\sb{\rm w}$ seemed to correlate well with bacterial growth (although a specific solute effect is expected). In a slow exchange (solid system, cellulose, or gums with added solute), mold germination and growth, as well as survival under extreme osmotic conditions, were found to be highly dependent on the molecular mobility of water. In such cases, a$\sb{\rm w}$ alone was a poor indicator. (Abstract shortened by UMI.)

Mesophilic cellulolytic Clostridium papyrosolvens C7 potentially could be used by industry for ethanol production from cellulose fermentation. The first goal of this study was to determine the pathways utilized by C. papyrosolvens C7 for the fermentation of cellulose and cellobiose, the soluble disaccharide product of cellulose hydrolysis. High-performance-liquid chromatography and gas chromatography analyses showed that acetate, ethanol, formate, lactate, malate, CO2, and H 2 were the end products of cellobiose fermentation by C. papyrosolvens C7. These products were quantified and a fermentation balance was calculated. Based on these analyses and the results of enzyme assays, a biochemical model for the fermentation pathways of C. papyrosolvens C7 is presented. It is suggested that formation of malate, a very uncommon fermentation product, served as an electron consuming reaction used to regenerate electron carriers (e.g., NAD+) during cell growth. In order to facilitate the metabolic engineering of C. papyrosolvens C7 for increased ethanol production and other properties desirable for industrial applications, a genetic exchange system involving the conjugative transposon Tn916 was developed. Tn916 was transferred from Enterococcus faecalis to C. papyrosolvens C7 in filter mating experiments. The tetM marker, conferring resistance to tetracycline, was used in the primary selection of transconjugants. Tn916 transfer frequencies ranged from 10 −7 to 10−5 per recipient. Highest transfer frequencies were obtained when recipient cells received a heat shock treatment, and both the donor and recipient cells were in the logarithmic phase of growth. A tetM gene probe hybridized to chromosomal DNA extracted from randomly selected C. papyrosolvens C7 transconjugants. These experiments indicated that Tn916 inserted into different sites on the chromosome of C. papyrosolvens C7. Tn 916 transposon mutagenesis was used to select low-acid-producing mutants. Fourteen mutant strains were selected by using the proton suicide method. Most of the mutant strains selected showed higher ethanol production. The third goal of this project was to determine the genome size of C. papyrosolvens C7. The restriction enzyme NotI was used to cleave intact C. papyrosolvens C7 DNA in agarose plugs. The resulting five DNA fragments were resolved by using pulse-field gel electrophoresis. The minimum size of the C. papyrosolvens C7 genome was determined to be 4.88 Mb.

The effect of water on growth and survival of Staphylococcus aureus was investigated using liquid (17O) and solid-state (1H) Nuclear Magnetic Resonance (NMR) spectroscopy. Different growth media, solute types, and methods of water activity (aw) adjustment (i.e. moisture versus solute variations) were studied. For the growth studies (>0.75 aw), mostly all water present was detected by the NMR and was highly mobile. Dependence of S. aureus growth was only partly dependent on the NMR signal intensity (amount of mobile or detected water) and partly dependent on the solute types. When aw was adjusted by changing moisture content, the use of brain heart infusion (BHI) or chicken meat media (CMM) did not affect the general conclusion. However, CMM resulted in an increased viscosity particularly at lower moisture content and partly influenced the NMR water mobility results. In general, it is postulated from this work that there is a critical amount of mobile water (based on 17O NMR intensity) of ∼40 g water detected/100 g sample below which S. aureus is significantly inhibited. For the survival study (<0.75 aw), the mobile proton signal was primarily due to the amount of water protons. Upon hydration, the onset NMR mobility increase also correlated with the monolayer value of water. A substantial increase in proton mobility (T2) was observed upon further increase in moisture content. Survival of S. aureus in a freeze-dried gum mixture was dependent on proton mobility, amount of mobile protons, and aw. Added mannitol and raffinose both protected the cells from osmotic-related death. The critical aw's at which cell death dramatically increased were in a similar aw range when proton mobility also increased. This suggested that molecular mobility facilitated the cell damage brought about by osmotic stress and in this case may serve as an indicator of water availability. Thus, molecular mobility plays a critical role in controlling cell survivability at a low moisture condition.

The relationship between physical characteristics and lipid oxidation was studied in model liquid and freeze-dried emulsions. A first part of the study involved the use of liquid emulsions consisting of linoleic acid, phosphate buffer, Tween-20 and sucrose. Iron sulfate and ascorbic acid were added to accelerate oxidation. The effect of variation in the system composition on oxidative behavior was investigated. Oxidation was influenced by the relative concentration of oil and emulsifier and their partition between different phases. When an excess of surfactant was present in the water phase, protection from oxidation was observed. Tween-20 competed with linoleic acid for catalysts and oxygen, and also, when in sufficient amount, provided a more compact coating of the interface. This prevented the contact between catalysts and oil. In the presence of sucrose, the increase in viscosity was expected to reduce oxidation. Although some protection was noticed, it did not correlated with the increase in sugar concentration. A protective action of sucrose other than viscosity, such as scavenging radicals and hydroperoxydes and/or quenching metals, was probably prevalent in this case. In the freeze-dried emulsions, maltodextrin and sucrose were added in equal amounts to provide a matrix. The effect of storage humidity on linoleic acid oxidation was studied. Lipid oxidation decreased after 32% relative humidity, contradicting the generally accepted oxidation theory that predicts a minimum in reaction rate at a water activity 0.3. Physical changes in the material counteracted the increased mobility and the increased reaction rate expected at the higher moisture. Structural collapse reduced oxygen availability and enclosed some of the linoleic acid that was previously exposed to oxidation. Moreover, sucrose crystallization caused some interface instability and consequent oil droplet coalescence. The coalescence of oil droplet's decreased the surface exposed to oxidation, contributing to the observed delay in oxidation at the higher relative humidity condition. Glass transition could not predict oxidative behavior in freeze-dried emulsions. The cooperation of the physical changes taking place in the system opposed the effect of glass transition on mobility. Although glass transition is often used to predict food stability, in some cases it may not be sufficient to accurately predict oxidative behavior. It is necessary to take into consideration other physical factors that may counteract its effect.

Food-processing by-products such as apple and cranberry processing wastes and fishery wastes were used in this study as the substrates for microbial bioconversion of value-added products such as beneficial fungal inoculants, for agriculture applications, food-grade enzymes for food processing applications, and phytochemicals for functional foods and food preservation. Three Trichoderma species, a Penicillium strain, and Rhizopus oligosporus were selected for producing beneficial bioinoculants from apple and cranberry processing wastes through solid-state fermentation. The effects of CaCO3, water, and NH 4NO3 on the growth of selected fungi were investigated, and the medium was optimized for maximum growth of each microorganism on apple and cranberry pomace. Fish protein hydrolysates were good supplementary nitrogen sources to fruit-processing wastes for microbial growth. The glucosamine content of fermented mixture was a good indicator of fungal biomass measurement during such heterogeneous solid-state fermentation. Both Trichoderma inoculants; and their water extracts produced from apple pomace significantly enhanced seed germination and seedling vigor in pea (Pisum sativum). They not only stimulated the seed germination and plant growth, but also increased the content of phytochemicals such as phenolics in seedlings. These results showed the potential of such bioinoculants in agricultural, food and pharmaceutical applications. Cranberry pomace was also a good substrate for bioconversion of food-grade enzymes such as β-glucosidase and phytochemicals such as phenolic acids by solid-state fermentation with a mushroom fungus Lentinus edodes . The enzyme had a low pH optimum and high stability at high temperature, thus has a potential applications in wine and juice processing for aroma and flavor enhancement. In bioremediation studies, a novel polymeric dye-degrading Penicillium species was isolated and identified. The isolate was able to aerobically decolorize both Poly R-478 and Poly S-119 in liquid systems. The decolorization involved initial mycelial adsorption of dye compounds followed by biodegradation through microbial metabolism, and it may be affected by medium constituents. Phytoremediation of polymeric dyes was also investigated with the use of several dye-tolerant oregano, thyme and rosemary clonal lines. As a result of natural defense mechanisms of plants, the total phenolics of most clonal lines decreased in response to polymeric dyes, whereas peroxidase activity increased. Stereomicroscopic observations revealed that the polymeric dyes were sequestered within the growing axis of the roots. Such polymeric dye-tolerant plants could be further targeted for developing pollutant tolerant rhizospheres that could accelerate microbial degradation of polymeric dyes and related aromatic compounds.

The physicochemical effects of pH, temperature, ionic strength and ingredient interaction on the formation of heat-denatured whey proteins and their resulting aggregation and gelation were investigated. Results were interpreted based on the molecular interactions that exist between protein molecules. Aggregation was dependent on protein concentration, pH, heat time and heat temperature. Optimal conditions for production of heat-denatured whey for use as a cold-set gelation ingredient were identified as 10 wt%, pH 7 and 75 to 85°C for 10 to 30 minutes, dependent on desired gel time and rheological properties. Whey protein aggregates were further characterized using Ultrasonic Attenuation Spectroscopy (UAS). UAS proved to be a valuable method for the investigation of molecular relaxation and scattering mechanisms in whey proteins. Electrostatic interactions proved crucial to cold-set gel network formation. Gel texture and optical properties were closely related to mineral content and type, with divalent cations inducing gelation via charge shielding and cross-linking, thereby reducing the amount of added salt necessary. Aggregation of heat-denatured whey proteins exhibited a concentration-dependent sensitivity to sucrose addition. Below 8 wt% sucrose network formation was retarded, as detected by suppression of rheological properties. This was attributed to the viscosity contribution by sucrose to the continuous phase, thereby reducing aggregate collision frequency. Above 8 wt%, the trend was reversed due to preferential dehydration of the protein molecules that encouraged protein-protein interaction. The addition of xanthan to a cold-set gelation system increased its textural properties. This was due to phase separation of the xanthan and heat-denatured whey proteins that resulted due to thermodynamic incompatibility. Excluded volume effects increased the effective concentrations of both biopolymers accounting for their resulting synergism. Finally, heat-denatured whey protein was added to an emulsion stabilized by non-ionic surfactant (Tween 20). Addition of salt caused aggregation of the proteins and was found to be dependent on protein and mineral concentration. A gel network formed around the non-interactive oil droplets to produce a thickened emulsion.

Dietary phenolic antioxidant compounds and the foods that contain them are emerging as an important new tool in the chemoprevention of various diseases associated with aging, such as diabetes mellitus and cancer. In the dissertation here, I discuss research performed to investigate novel approaches to harnessing the phenolic antioxidants found in soybean involving solid-state bioprocessing by dietary fungus such as Rhizopus oligosporus and Lentinus edodes (Shitake mushroom) and dark-germination sprouting to promote phenolic synthesis. Further, I discuss important new biofunctionalities discovered for dietary phenolic antioxidants from soybean, herbs, and other foods that may have implications for modulation of diabetes mellitus and associated hypertension, as well as for combating troublesome food-borne or diet-associated bacterial pathogens such as stomach cancer and ulcer-linked Helicobacter pylori and the causative agent of listeriosis, Listeria monocytogenes.

The aim of this dissertation research was to develop innovative strategies to enhance biological functionality and improve consistency of phenolic phytochemical profiles in cranberry products and byproducts. Solid-state bioprocessing (SSB) of cranberry pomace with food grade fungi Rhizopus oligosporus and Lentinus edodes was used to mobilize phenolic antioxidants within a consistent phytochemical profile and improve biological functionality. The effect of SSB on antimicrobial activity was tested against three important food-borne pathogens Listeria monocytogenes , Vibrio parahaemolyticus and Escherichia coli O157:H7. Further, the antimicrobial activity of the extracts was also tested against gastric cancer and gastric ulcer-linked Helicobacter pylori. The results indicated that SSB mobilized functionally important phenolic phytochemicals such as ellagic acid and enhanced antioxidant activity. SSB also resulted in enhancing the antimicrobial activity of the extract. The results provided an insight into the mechanism of antimicrobial activity of phenolic phytochemicals. Cranberry synergies with functional biphenyls ellagic acid and rosmarinic acid were designed for enrichment of antioxidant, anti-H. pylori, antimutagen and DNA protection properties. Results indicated that cranberry synergies with biphenyls improved their antioxidant activity. The antimutagenic and DNA protective functions properties of cranberry synergies were found to be significantly higher than pure compounds. Cranberry synergies linked to enhanced antioxidant activity and phytochemical profile also improved the antimicrobial property of the extract against gastric ulcer-linked H. pylori. The anti-H. pylori activity of cranberry was further enhanced by synergistically blending cranberry extract with other fruit and herb extracts. The mechanism of action of these cranberry synergies with biphenyls on modulating the antioxidant enzyme response was investigated in germinating fava bean sprouts and oxidatively stressed porcine muscle tissue. From this investigation phenolic antioxidants from plants appear to mediate their biological functionality by modulating antioxidant systems in eukaryotes by more than one mechanism. These functions were carried out either as free radical scavenging antioxidants and more importantly by inducing antioxidant enzyme responses in the cellular systems. The results also indicated that pure biphenyls functioned more efficiently when they were in a cranberry background. The results provide an important insight into the possible mechanism of action of fruit phytochemicals in biological systems and also showed that they can be improved in synergy with specific biphenyls. (Abstract shortened by UMI.)

Oil-in-water emulsions (hexadecane, minimal media (M9) and Tween 20) were used as a model system to study the growth of E. coli O157:H7 ATCC 43895. Stationary phase cell density decreased as the hexadecane concentration was increased (0%, 5%, 20%, and 40% (w/v)) and biphasic growth was observed in 40% emulsions supplemented with 0.4% glucose. Thin aggregate fimbriae (curli) were observed using SEM, and a greater percentage (P < 0.001) of curli-producing colonies were isolated from 40% emulsions. Heat resistance (55°C and 58°C) of emulsion-grown cells was greater than broth-grown cells, indicating that growth and physiology under heterogeneous conditions differ from cells grown in liquid. In addition, the curli expression and biofilm formation of this pathogen in a low nutrient environment was investigated. The results showed that the curli expression in this O157:H7 strain was temperature independent and more stable under low nutrient conditions. This organism was able to form biofilm on a PVC surface when grown in M9 but not LB broth. There was a positive correlation coefficient between biofilm formation and curli expression in this strain at 37°C (p < 0.05). Biofilm formation increased as curli production increased. These findings suggest that the ability of E. coli O157: H7 to express curli and produce biofilm in a low nutrient environment may contribute to the surface growth in trough water in cattle farm and serve as a source for recontamination. The rpoS mutant of O157:H7 43895 was used to study the impact of rpoS on heat tolerance, curli production and biofilm formation. The rpoS gene encoding a sigma factor (δ s) is a central regulator for several stationary phase and stress response inducible genes in E. coli including heat resistance, curli expression and biofilm formation. The results presented here indicate that rpoS regulated proteins were being expressed in cells grown in heterogeneous system and were responsible for enhanced heat resistance. The curli expression requires rpoS regulation. However, higher biofilm formation in the rpoS mutant was observed with no evidence of curli expression indicating that factors additional to rpoS regulated proteins and curli expression may have influenced biofilm formation in this O157:H7 strain.