<p> The objective of this study was to determine the influence of market type and sampling time on <i>Salmonella</i> and <i>Listeria </i> prevalence and microbiological quality of 540 beef, pork, and whole chicken samples collected in 6 supermarkets (SM), 6 indoor markets (IM), and 6 open markets (OM) at opening (T0) and 4 h after the opening (T4) in Vietnam. <i> Salmonella</i> and <i>Listeria</i> prevalence ranged from 30.4 to 71.0% and 56.6 to 99.9 %, respectively, in beef, pork, and chicken in Vietnam. Aerobic bacteria counts ranged from 10.5 to 11.6 log CFU/g, whereas, <i> E. coli</i> and coliform counts ranged from 7.2 to 11.4 log CFU/g in beef, pork, and chicken in Vietnam. <i>E. coli</i> counts were influenced by the interaction of market type and sampling time in beef and pork. Market characteristic data that were considered relevant to microbiological safety of fresh meat and poultry products were collected for individual samples. </p>

Chronic oxidation linked diseases are on a rise and are one of the leading causes of death globally. Epidemiological evidence increasingly points towards consumption of fruits and vegetables as a preventive way to manage early stages of chronic oxidation linked diseases. Oxidation linked diseases are caused by excessive reactive oxygen species (ROS) generated by a disruption in cellular antioxidant homeostasis due to an overload of calories combined with stress, no excerise and a diet low in antioxidants. Phenolic compounds can not only act as antioxidants but also stimulate the activities of antioxidants enzyme through protective pathways which can help modulate cellular protection. The aim of this dissertation was to use probiotic fermentation to enhance the phenolic and antioxidant compounds in fruit systems which can form the basis of functional food design. The potential of these food systems for disease prevention was investigated in eukaryotic systems through understanding the role of critical metabolic pathways involed in prevention of oxidation linked chronic diseases. Based on structure-function rationale, antioxidant, anti-hyperglycemia and anti-hypertensive potential of phenolic compounds in tea and the effect of extraction time and different degrees of fermentation were investigated in in vitro models. Results indicated that the most fermented teas and a longer extraction time had the highest potential. Further these extracts also had higher H. pylori inhibition potential. Probiotic fermentation of fruit juices with L. helveticus was used to mobilize phenolics and improve biological functionality by maintaining a consistent phytochemical profile. Results indicated that total phenolic and antioxidant potential decreased with feremnetation. However &agr;-glucosidase inhibitory activity and H. pylori inhibitory potential increased with fermentation. Investigation into the mechanism of H. pylori inhibition with fermented cherry extracts revealed inhibition of proline dehydrogenase as the likely mode of action. The potential of fermented apple extracts was further investigated as a phytochemical elicitor in eliciting phenolic and antioxidant response in germinating fava bean. The results indicated a stimulation of phenolic and antioxidant response likely through the stimulation of carbon flux through glycolytic pathways. In yeast, fermented apple extracts accelerated cell death in the presence of peroxide stress in pretreatment model whereas it provided protection against oxidative stress and prevented cell death in concurrent model. Chitosan oligosachharide treatment was investigated as a potential replacement of cancer causing diphenylamine treatment for scald reduction in Cortland apples. Although the treatment did not have any effect on scald reduction, it provides better protection in storage by stimulating phenolic and antioxidant response which related to better health relevant functionality.

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.)

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.

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.

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.

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.

The food-borne pathogen Listeria monocytogenes can attach to environmental surfaces and form biofilms which can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. The overall objective of this study was to identify the genetic requirements of biofilm formation by L. monocytogenes. In-frame deletion mutants of a putative mutarotase gene (lmo2476/lin2619 ) were constructed to investigate its influence on Listeria biofilm formation. No biofilm phenotype changes were observed between the wild type and the corresponding mutants, indicating that the putative mutarotase gene was not involved in Listeria biofilm formation under the conditions tested. A mariner-based transposon mutagenesis was performed to generate mutants of L. monocytogenes. A mutant library consisting of 6,500 colonies was screened for reduced biofilm formation. A total of 24 distinct loci were identified, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation of L. monocytogenes. A putative DNA translocase gene, lmo1386, was further characterized. The mutant was complemented, and the complemented mutant restored its biofilm phenotype. The lmo1386 mutants showed reduced initial attachment abilities, and had higher numbers of elongated cells when grown in a nutrient TSBYE broth. However, the exact mechanisms of how lmo1386 affects biofilm formation remain to be elucidated. The inhibitory effects of EDTA against biofilm formation of L. monocytogenes were investigated. EDTA at a concentration of 0.1 mM efficiently inhibited biofilm formation of L. monocytogenes without affecting its planktonic growth. EDTA functions in the early stage by affecting the initial attachment of L. monocytogenes cells to surfaces, though the mechanisms remain unclear. The role of extracellular DNA (eDNA) in the formation of L. monocytogenes biofilm was determined indirectly by treatments of DNase I. Our data adds to the knowledge that eDNA plays an essential role in attachment and maintenance of L. monocytogenes biofilm. The pre-formed biofilms on the wells of microtiter plates could be efficiently removed by DNase I, suggesting a potential use of DNase I to eradicate the existing L. monocytogenes biofilms.

Vibrio vulnificus is a Gram-negative estuarine bacterium that can cause wound infection, primary septicemia and gastroenteritis in susceptible individuals. Since it is a notably lethal pathogen associated with seafood, rapid detection and quantification of V. vulnificus is an important issue for the protection of consumer health and the seafood industry. With conventional PCR the minimum detection level with a new nucleic acid dye (GelStar stain) was 16 CFU per PCR reaction for pure culture compared to 40 CFU with ethidium bromide (EB). With real time PCR, the detection limit was 100 CFU/g of tissue with a linear detection range of 102 to 108 CFU/g without enrichment and 1 CFU with a detection range of 1 to 106 CFU/g of tissue after 5h enrichment. A 508-bp DNA fragment was used as an internal standard for competition with target DNA in competitive PCR, by which the detection sensitivity was 220 CFU of pure culture per PCR reaction, 2700 CFU/PCR with shellfish tissue without enrichment, and 7 CFU/PCR with shellfish tissue after 10 h enrichment. Geobacillus stearothermophilus and hemoglobin were used as a reference strain and a PCR inhibitor respectively, to establish comparative real time PCR, by which the minimum detection sensitivity was 102 CFU per ml of pure culture and 103 CFU per gram of clam tissue. The equation (Ct, T0 = Ct,TX - ΔCt) can be used for enumeration of V. vulnificus in tissue in conjunction with a standard curve relating Ct values to Log genomic targets in the absence of PCR inhibitors via comparative RT-PCR. Amplification of DNA from heat killed cells in a mixture with viable cells was successfully inhibited by treatment of the cells with 2.5 μg/ml of ethidium bromide monoazide (EMA) followed by 15 min photolysis, whereas the DNA from viable cells present was successfully amplified by real time PCR. Using this technique, the detection sensitivity for viable bacterial cells was 10 CFU per ml of pure culture and 100 CFU/g of clam tissue.

An $\alpha$-amylase (EC 3.2.1.1) secreted by Clostridium perfringens NCTC 8679 Type A was purified to homogeneity and characterized. It was isolated from concentrated cell-free culture medium by ion exchange and gel permeation chromatography. The pH optimum and pI of the enzyme were 6.5 and 4.75 respectively. The estimated molecular weight of the purified enzyme was 76 000. Calcium (5 mM) increased the optimal temperature for activity of the purified enzyme from 30 to 40$\sp\circ$C. The purified enzyme was inactivated between 35 and 40$\sp\circ$C which increased to between 45 and 50$\sp\circ$C in the presence of calcium. The purified enzyme produced a mixture of oligosaccharides as major endproducts of starch hydrolysis indicating $\alpha$-amylase activity. The effect of glucose and other sugars on sporulation and extracellular amylase production by Clostridium perfringens NCTC 8679 type A in a defined medium was also studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity while disaccharides such as lactose, maltose and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10 mM except for fructose (25 mM). Levels of heat resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The extracellular amylase produced by C. perfringens exhibited eight major extracellular amylolytic activity bands when examined by activity staining following polyacrylamide gel electrophoresis (PAGE). Extracellular amylolytic enzymes released in sporulation process were different from those released during vegetative growth (extracellular amylase accumulation) whereas their intracellular amylolytic activities remained identical. A significant increase of an endo-acting amylolytic activity which cleaved both $\alpha$-1,4- and $\alpha$-1,6-glucosidic linkage was observed when C. perfringens sporulated. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in C. perfringens whereas regulation of amylolytic activity during sporulation is possibly mediated by sporulation-related, amylolytic enzyme modification.