Listeria sp. can grow on surfaces and be a member of multi-species biofilms within food processing plants, rendering them more resistant to sanitizers and other antimicrobial agents. Little is known of the cellular physiological processes involved in Listeria sp. growth on surfaces. The purpose of this study was to generate random transposon mutants in order to identify genetic determinants contributing to biofilm formation. Listeria innocua ATCC 51742 serotype 4b was selected for this study since it produces high levels of biofilm in a laboratory assay. Transposon mutagenesis was performed using plasmid pLTV3 (Tn917) with a temperature sensitive origin of replication. Mutants were selected by growth in the presence of erythromycin and lincomysin at 41°C. A library, consisting of 2500 mutants, was further screened by use of a PVC microtiter plate assay at 32°C at low nutrient levels. Reduced surface growth (RSG) mutants were defined as having a mean stained biofilm at 2 std dev below the mean of the wild-type strain. A total of 36 RSG mutants were isolated and further characterized by testing cellular motility and initial adhesion phenotypes. RSG mutants exhibited various phenotype profiles, suggesting a number of genes involved in biofilm formation of L. innocua. RSG 24 mutant was further characterized, showing an increased negative surface charge. RSG 24 mutant scanning electron microscopy and epifluorescence microscopy results showed reduced surface growth area coverage compared to wild-type. An arbitrary PCR, PCR product cloning and sequencing revealed that Tn917 transposition took place in lin2619, a gene homologous to mutarotase. Surface growth characteristics were restored to RSG 24 by complemetation of the lin2619 mutarotase gene in trans to levels comparable to the wild-type strain. Complementation in trans and results indicating altered cell surface characteristics, our study indicates that lin2619 is involved in L. innocua adhesion and surface growth.

Lipids such as omega-3 fatty acids and citrus oils are important food components due to their nutritional and flavor benefits, respectively. Both of these food lipids are susceptible to chemical degradation resulting in formation of unacceptable off-flavors, nutrient and flavor loss. Oil-in-water emulsions could be used as an effective tool to protect these chemically sensitive lipids from deterioration by engineering the emulsion droplet interfacial membrane (e.g. charge and/or thickness) using proteins or multiple layers of emulsifiers. The objective of this study was to investigate the effectiveness of the interfacial characteristics of emulsions stabilized with proteins or multiple layers of emulsifiers in preventing oxidative deterioration of omega-3 fatty acids and citrus oil flavor compounds. Factors influencing the stability of protein-stabilized oil-in-water emulsions at pH 3.0 were examined. Physical instability of whey protein isolate (WPI)-stabilized corn oil-in-water emulsions increased considerably after thermal processing at ≥ 70°C in the presence of ≥ 150 mM NaCl. WPI-stabilized oil-in-water emulsions containing 25 wt% algal or menhaden oils at pH 3.0 had good oxidative stability when pasteurized and stored at 4°C. The addition of EDTA and mixed tocopherol isomers further increased oxidative stability. These results suggest that WPI-stabilized algal or menhaden oil-in-water emulsions at pH 3.0 containing EDTA may be used as an ingredient delivery system to incorporate ω-3 fatty acids into functional foods. Citral/hexadecane- or limonene/hexadecane-in-water emulsions were prepared with varying interfacial properties by coating the oil droplets with WPI, sodium dodecyl sulfate (SDS), SDS-chitosan or gum arabic (GA). Emulsifier type had little influence on the rate of citral degradation. However, WPI and SDS-chitosan were more effective at retarding the oxidation of citral degradation products and limonene in comparison to emulsions stabilized by GA. Inhibition of oxidation by the WPI or SDS-chitosan systems could be due to the ability of these emulsifiers to produce cationic emulsion droplet interfacial membranes that inhibit the interactions between dispersed lipids and continuous phase prooxidants such as iron. This research may lead to development of new strategies to improve the stability of emulsified products containing citrus oils.

This study focuses on applying Nuclear Magnetic Resonance (NMR) to differentiate heterogeneous domains of starch Mobility and distribution of water and starch chains in cassava starch from crops grown in drought and rainy reasons, harvested at 6 months and 12 months, were investigated by deuterium (2H) solid state NMR and proton (1H) spin-spin relaxation time (T 2) distributions. Changes in water and starch chain mobility with heating, freezing, and acid hydrolysis were also studied. In a very low moisture range (<5% dry basis), all water present was immobile (rigid) in both drought and rainy cassava (A-type crystals), and potato starches (B-type crystal). Thus, the amount of bound water was irrespective of degrees of crystallinity and crystal types. The “Monolayer” value estimated from water sorption isotherms (7–9% dry basis) over-estimated the level of bound water in starch (when compared with NMR data). With increasing water content, T, decreased to a minimum at a moisture range of 14–17% (dry basis) marking its onset of molecular plasticization of the starch chains. Packed beds of starch granules (54% water, dry basis) were subjected to heating and freezing treatments. NMR T2 distributions of water and starch protons were characterized with a continuous model. Intragranular water (T2 ∼ 3 ms, disappeared below 0°C) and extragranular water (T2 ∼ 20 ms) were observed. An extra long T2 (∼100 ms) population (postulated as water associated with leached amylose) was developed upon gelatinization. The swelling and melting of starch from the drought crop were shifted to a higher temperature than those from the rainy crop resulting from a possible closer packing of rigid amylopectin and/or amylose chains. Upon gelatinization, rigid amylopectin (T2 ∼ 10 μs) disappeared, while the mobile amylopectin (in the amorphous region of semicrystalline lamellae, T2 ∼ 1 ms) increased. An additional fraction with very long T 2 (∼50 ms) emerged at 65°C and above indicating an increase in mobile/soluble starch. This study demonstrated a possible use of NMR to monitor changes in starch chains and related water mobility in intact starch granules, which is a unique feature not offered by other methods.

The main objective of this research was to better understand the formation, stability and properties of emulsions having lipid nanoparticles with tunable functional properties by controlling the composition and structure of the biopolymer interface, in order to develop better food-grade delivery systems. Initially, the influence of environmental stresses (pH, heating and salts) on the physicochemical properties of cationic lactoferrin (LF)-stabilized oil-in-water emulsions was investigated. At ambient temperature, the emulsions were found to be stable at all times except when pH was close to pI. When LF-coated droplets were heated in distilled water, and then their pH was adjusted in the range 2 to 9, they were highly unstable to aggregation at pH 7 and 8. These results have important implications for the formulation and production of emulsion-based products using lactoferrin as an emulsifier. Next, we studied the properties and stability of multilayer emulsions formed using the primary emulsifier lactoferrin and secondary polysaccharides like low methoxyl pectin (LMP), high methoxyl pectin (HMP) and alginate. At neutral pH, electrostatic attractions occurred between the anionic groups on the polysaccharide molecules and the cationic patches on the protein surfaces. In the absence of polysaccharide, the LF-coated droplets were highly unstable to aggregation when heated above about 60 ºC at pH 7, presumably because thermal denaturation of the adsorbed proteins increased droplet attraction. Changes in the physicochemical properties and digestibility of both the primary LF and the secondary LF-polysaccharide emulsions, under simulated gastrointestinal conditions were monitored. The presence of a dietary fiber coating around the initial lipid droplets had little influence on the total extent of lipid digestion in simulated intestinal fluid (SIF), but LF-alginate emulsions had a slower initial digestion rate than the other emulsions. These results suggest that the dietary fiber coatings may become detached in the small intestine, or that they were permeable to digestive enzymes. Pepsin was found to have little influence on the physical stability or digestibility of the emulsions. Next, we fabricated emulsions with oil droplets coated by sequential electrostatic deposition of cationic LF and anionic β-lactoglobulin (BLG) at pH 6.5: LF, LF-BLG, LF-BLG-LF, and LF-BLG-LF-BLG. Changes in the physicochemical properties of these systems were characterized when they were exposed to environmental stresses and simulated small intestine conditions. LF-coated droplets were stable throughout the entire pH range which was attributed to strong steric repulsion. All the nanolaminated droplets were unstable to aggregation at pH 5, which is between the isoelectric points of BLG and LF. Finally, a "premix" approach was utilized to fabricate interfacial coatings around the lipid droplets, instead of the LbL approach. This method involved mixing BLG and LF prior to emulsion formation and the influence of environmental stresses on the properties of these emulsions was examined. Droplets coated by BLG were unstable to aggregation near their isoelectric point (pH ≈ 5), whereas those coated by LF were stable across the whole pH range. The stability of emulsions to pH induced aggregation improved as the ratio of LF-to-BLG in the mixed systems was increased. Lipid droplets coated by either LF or BLG were unstable to aggregation at high salt concentrations (500 mM NaCl, pH 6.5), but those stabilized by mixed protein coatings (LF and BLG) were stable, which was attributed to an increase in interfacial thickness and steric repulsion. Droplets coated by BLG were stable to droplet aggregation after thermal treatment (30 to 90 oC, 0 mM, NaCl pH 7), whereas those coated by LF were highly unstable when heated above their thermal denaturation temperature. The thermal stability of the droplets decreased as the amount of LF in the mixed systems increased. (Abstract shortened by UMI.)

The objective of this work was to study parameters involved in the transfer of Listeria monocytogenes from surfaces to foods and vice versa. We evaluated the influence of surfaces (stainless steel and high density polyethylene, HDPE), inoculation method (biofilm growth and attached cells), hydration levels (dry and wet), and foods on listerial transfer. A four L. monocytogenes strain cocktail was used to inoculate either processing surfaces or foods as growing biofilms or attached cells. Transfer experiments were performed at a constant contact time (30 s) and pressure (45 kPa). After transfer, cells transferred to surfaces or foods were counted, and the efficiency of transfer (EOT) calculated. Our results showed that when calculating EOTs, data were normalized and the initial inoculation level had no effect on the proportion transferred (P>0.05). Material type showed to be a significant factor, with greater numbers of Listeria transferring from bologna to stainless steel (EOT=0.49) when compared to polyethylene (EOT=0.28, P<0.01). When transfer was conducted from inoculated surfaces to uninoculated food, our results strongly suggested that stainless steel surfaces (EOT=0.52) transferred more L. monocytogenes to foods than HDPE (EOT=0.21, P=0.05). Regarding the inoculation method, biofilms tended to transfer more Listeria to foods (EOT=0.57) than attached cells (EOT=0.16). Transfer from inoculated surfaces to foods was significantly higher for dried biofilms growing on stainless steel (EOT=1.5, P<0.05). The effect of hydration level was further evaluated by equilibrating biofilms over saturated salt solutions at 20°C for 24 h (94%, 75%, 58% and 33% relative humidity) prior to transferring. Our work showed that as biofilms become drier, the listerial transfer increased significantly (P<0.05). We hypothesize that weakened cell-to-cell and cell-to-surface interactions of biofilms upon drying increases transfer to food products. With the use of AFM we determined that the adhesion forces between colloidal (glass and polyethylene) probes and listerial biofilms. The results showed that the maximum adhesive force to polyethylene (-113.38 nN) was significantly higher than that of glass (-85.42 nN, P<0.001). Sanitation practices in food industry must be addressed to avoid biofilm formation. Once biofilms are formed, they are harder to remove and listerial transfer and potential cross-contamination increases.

Abstract not available

The combination of water and surface active compounds found naturally in commercially refined vegetable oils have been postulated to form physical structures known as association colloids. This research studied the ability of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC) and water to form physical structures in stripped soybean oil. Interfacial tension and fluorescence spectrometry results showed the critical micelle concentration (CMC) of DOPC in stripped soybean oil was 650 and 950 µM, respectively. Light scattering attenuation results indicated that the structure formed by DOPC was reverse micelles. The physical properties of DOPC reverse micelles were determined using small-angle X-ray scattering (SAXS) and fluorescence probes. These studies showed that increasing the water concentration altered the size and shape of the reverse micelles formed by DOPC. The impact of DOPC reverse micelles on the lipid oxidation of stripped soybean oil was investigated by following the formation of primary and secondary lipid oxidation products. DOPC reverse micelles had a prooxidant effect, shortening the oxidation lag phase of SSO at 55 °C. It also was not able to change the lipid oxidation of stripped soybean oil compared with DOPC reverse micelles at same concemtration ( i.e., 950 µM). 1,2-dibutyl-sn-glycerol -3-phosphocholine (DC4PC) which has the shorter fatty acid than DOPC was not able to form association colloids and did not impact lipid oxidation rates. This indicated that the choline group of the phospholipid was not responsible for the increased oxidation rates and suggested that the physical structure formed by DOPC was responsible for the prooxidant effect. The impact of the DOPC reverse micelles on the effectiveness and physical location of the antioxidants, α-tocopherol and Trolox was also studied. Both non-polar (α-tocopherol) and polar (Trolox) were able to inhibit lipid oxidation in stripped soybean oil in the presence of DOPC reverse micelles. Trolox was a more effective antioxidant than α-tocopherol. Fluorescence steady state and lifetime decay studies suggested that both α-tocopherol and Trolox were associated with DOPC reverse micelle in bulk oil. Trolox primarily concentrated in the water pool of reverse micelle since it quenched NBD-PE fluorescence intensity with increasing concentrations. A portion of α-tocopherol was also associated with the aqueous phase of the DOPC reverse micelles but this was likely at the oil-water interface since α-tocopherol is not water soluble. The addition of ferric chelator, deferoxamine (DFO) to stripped soybean oil significantly prevented the lipid oxidation caused by DOPC reverse micelles as the lag phase was extended from 2 to 7 days. DFO was also found to increase the antioxidant activity of both Trolox and α-tocopherol. Trolox and α-tocopherol were found to be rapidly decomposed by high-valence Fe(III) while low-valence-state (Fe (II) was much less reactive. Fe(III) was also consumed by both hydrophilic Trolox and lipophilic α-tocopherol presumably though reduction to Fe (II). DOPC reverse micelles were able to decrease antioxidants-iron interactions as evidence by a decrease in antioxidant depletion by iron and a decrease in iron reduction by the antioxidants. These results suggested that the ability of DFO to increase the antioxidant activity of α-tocopherol and Trolox was due to its ability to decrease free radical production and not its ability to decrease direct iron-antioxidant interactions. Overall, the results presented in this dissertation show phospholipids and water can form reverse micelles in edible oils. These reverse micelles increase lipid oxidation rates by increasing the prooxidant activity of iron. Free radical scavenging antioxidants can inhibit oxidation promoted by the reverse micelles with polar Trolox being more effective than non-polar α-tocopherol presumably because Trolox is more highly associated with the reverse micelle. The reverse micelles produced by DOPC protected α-tocopherol and Trolox from direct degradation by iron. The knowledge gained from this study will improve our understanding of the mechanism of lipid oxidation in bulk oils which will hopefully provide new technologies to improve the oxidation stability of edible oils. For example, it may be able to use oil refining technologies to remove prooxidative minor components that for physical structure in bulk oils.

Peroxynitrite (ONOO−), formed from the nearly diffusion limited reaction between nitric oxide and superoxide, could be an important prooxidant in muscle foods. The objective of this dissertation was to (1) determine whether peroxynitrite caused oxidation of muscle lipids; (2) assess the potential for nitric oxide formation in muscle; and (3) examine the utility of 5-nitro-γ-tocopherol (NGT) as a peroxynitrite biomarker in muscle foods. Exogenous peroxynitrite induced oxidation of liposomes and skeletal muscle microsomes and homogenates. Skeletal muscle homogenate oxidation by reagent peroxynitrite was suppressed by metal chelators. Fresh muscle exhibited nitric oxide synthase (NOS) activity and thus the potential to produce peroxynitrite. NOS activity was reduced during refrigerated storage and by one freeze/thaw cycle. NOS activity in muscle was not affected by pH over the range of 4.5–7.4, was inhibited by 1 and 2% NaCl, and was stimulated at internal cooking temperatures up to 55°C but was completely inhibited at higher temperatures. NGT formation was induced by exogenous peroxynitrite in turkey and chicken muscle homogenates, but turkey and chicken muscle did not produce detectable levels of NGT during storage. Results of this dissertation indicate that peroxynitrite can cause lipid oxidation in muscle lipids and that post-mortem muscle has the potential to produce endogenous peroxynitrite via the activity of NOS. However, NOS was only active for the first several days of postmortem storage and no detectable NGT was observed in turkey and chicken during storage. This suggests that either no peroxynitrite is formed in post-mortem muscle or that low levels of peroxynitrite are produced which limit the detectablility of NGT using the methods employed here.

Abstract not available

Abstract not available