No description available.

Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the <u>M</u>ammalian <u>C</u>ell-based <u>I</u>mmuno<u>A</u>ssay (MaCIA) was validated with <i>Salmonella enterica</i> serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium,11 other <i>Salmonella</i> serovars, and 14 non-<i>Salmonella</i> spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1-10 CFU/25 g was 16-21 h using a traditional enrichment set up but the detection time was shortened to 10-12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.

<p>Retail environments are critical transmission points for <i>Listeria monocytogenes</i> to humans. Past studies have shown <i>L. monocytogenes</i> contamination varies widely across retail environments. <i>L. monocytogenes</i> can transmit among environmental surfaces and subsequently from environment to food via cross-contamination. Modified SSOPs (sanitation standard operating procedures) have been shown to have limited impact on reducing <i>L. monocytogenes</i> prevalence in retail deli environments. Food safety culture and climate, such as beliefs, values, and hygiene behaviors, have been identified as factors impacting food safety performance and microbial outputs. Handwashing and its compliance are among the most prominent personal hygiene aspects subjected to investigation in the past decade, illustrating hygiene behavior as a risk factor and an important consideration to ensure food safety. Additionally, effective management and well-designed infrastructure, such as vertical and lateral communication, employees’ training, accountability, and equipment designed to prevent cross-contamination, have also been described as critical contributors to a sustainable food safety program. However, given such a deadly foodborne pathogen as <i>L. monocytogenes</i>, the correlation between food safety culture and its prevalence remains unknown. We hypothesized that there was a relationship among food safety culture management, infrastructure, and <i>L. monocytogenes</i> prevalence at retail. Our goal is to identify additional risk factors on <i>L. monocytogenes </i>control, develop feasible recommendations, and direct resources to enhance food safety. </p> <p>In the present dissertation, we developed and implemented a predictive risk model, along with employee- and management-implemented SSOPs, in 50 deli establishments across six U.S. states to evaluate and control <i>L. monocytogenes</i> contamination risk and prevalence (Chapter 2). The predictive risk model, based on logistic regression, uses five environmental sites to predict <i>L. monocytogenes</i> prevalence in the entire deli environment. It identified 13 high-risk stores, seven of which were confirmed during subsequent monthly sampling. We found that deep clean intervention reduced <i>L. monocytogenes </i>prevalence on non-food contact surfaces both immediately after the intervention and during follow-up, with marginal significance (α_adj=0.0125). The employee- and management-implemented deep clean can control <i>L. monocytogenes</i> prevalence in retail delis; the predictive risk model, though conservative, will require further validations and can be useful for surveillance purposes. </p> <p>Complementary to the above study, we tackled the <i>L. monocytogenes</i> challenge via food safety culture and climate approach (Chapter 3). Concurrently to the monthly environmental sampling, we distributed food safety culture and climate survey to the 50 stores, with one manager and up to five associates from each establishment, over a 12-month period and overlapped with before, after, and follow-up deep clean. We found that stores with lower <i>L. monocytogenes</i> contamination risk had better food safety culture, including greater sense of commitment to food safety program (p_adj=0.0317) and more complete training (p_adj=0.0117). Deep clean improved managers’ (p_adj=0.0243) and associates’ (p_adj=0.0057) commitment to food safety. This study indicates that food safety culture and climate are crucial component in building a viable, sustainable food safety program. </p> <p>Another survey tool was used to evaluate infrastructure designs, management strategies, and sanitation practices in relation to <i>L. monocytogenes</i> control in retail produce environments (Chapter 4). We distributed the survey to 30 retail produce departments across seven U.S. states. Hand hygiene, minimizing cross-contamination, and maximizing equipment cleanability were the most prominent factors in <i>L. monocytogenes</i> control.</p>

<div><div><div><p>Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, and biofilms are known to be the frequent source for infection and outbreaks of foodborne illness. Therefore, it is essential to understand the pathogenicity of bacteria in biofilms and methods to inactivate biofilm-forming microbes from food processing environments, including school cafeteria or other community-based food production facilities, and to prevent foodborne outbreaks. Pathogen transmissions occur primarily through raw or under cooked foods and by cross contamination during unsanitary food preparation practices. Then, pathogens can form biofilms on the surface and become persistent in food production facilities and can be a source for recurrent contamination and foodborne outbreaks. In this study, our first aim was to use L. monocytogenes as a model pathogen to study how an enteric infectious pathogen isolated from biofilm modifies its pathogenesis compared to its planktonic counterpart. Both clinical and food isolates with different serotypes and biofilm-forming abilities were selected and tested using cell culture and mouse models. L. monocytogenes sessile cells isolated from biofilms express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains revealed that at 12 and 24 h post-infection (hpi), L. monocytogenes burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile L. monocytogenes from intestinal content are about 6.0- and 280-fold higher than the sessile inoculum, respectively, suggesting sessile L. monocytogenes can still upregulate virulence genes shortly after ingestion (12 h).</p><p>After learning biofilm isolated L. monocytogenes cells have similar virulence potential as the planktonic counterparts, our next goal was to effectively prevent or inactivate biofilms using food-grade natural microbials. Since L. monocytogenes cells are usually found in multi-pathogen biofilm in nature, I combined two food-grade broad-spectrum natural antimicrobials, chitosan nanoparticles (ChNP) and ε-poly-L-lysine (PL), as ChNP-PL nanoconjugates and tested its function on single or mixed culture biofilms of L. monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Enteritidis, and Pseudomonas aeruginosa. ChNP- PL not only was able to significantly (P<0.05) prevent the biofilm formation but also inactivate pre-formed biofilms when analyzed by crystal violet staining and plate counting. In vitro cytotoxicity analysis (LDH and WST-based assays) using an intestinal cell line, indicated ChNP- PL to be non-toxic. In conclusion, our results showed ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens in food processing environment. Application of ChNP-PL could inhibit the colonization of foodborne pathogens, minimize cross-contamination during food production, and eventually reduce foodborne outbreaks.</p></div></div></div>

<div>Fresh produce has been the leading source of foodborne illness outbreaks in the US, surpassing typical pathogen carriers such as meat, dairy, and seafood. Among the fresh produce popular to the consumers, cantaloupe and sprouts are mostly susceptible to pathogen contaminations and outbreaks. However, it has been a challenge to address the key factor in the contamination - the biofilms formed by pathogens are highly resistant to conventional washing and cleaning procedures. For cantaloupe, the net-like and porous surface forms a barrier for washing. For sprouts, the fragile texture of seedlings prevents aggressive cleaning operation and biofilm removal.</div><div><br></div><div>In this study, innovative interventions were developed to improve microbial safety of fresh produce, using cantaloupe and alfalfa sprouts as models. For cantaloupe, abrasive brushing was designed to remove pathogen biofilm from cantaloupe. Our research found pathogens could form biofilm at cantaloupe rind surface as the residence time of pathogens increased. Biofilm formed on cantaloupe rind was imaged by cryo-scanning electron microscopy (cryo-SEM), and its resistance to sodium hypochlorite and lauroyl arginate ethyl (LAE) was confirmed. Furthermore, abrasive brushing with peroxyacetic acid (PAA) could effectively remove biofilm formed at cantaloupe rind. The efficacy of this novel cleaning technique was highly desirable, which could achieve 3 log reduction in pathogen population. Mechanism of abrasive brushing to remove biofilm at cantaloupe rind surface was also proposed. Conceivably, brushing with diatomaceous earth (DE) and PAA could be an innovative and cost-effective method to remove pathogen biofilm from cantaloupe rind.</div><div><br></div><div>For alfalfa sprouts, since most of the outbreaks are linked to the sprouting seeds, seed disinfection treatments are considered to be the most effective method to improve microbial safety of sprouts. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. The calcium alginate coating in the presence of 2.5% lactic acid (CA-LA coating) reduced foodborne pathogens inoculated on alfalfa seeds to an undetectable level on day 1 during 28 day-seed storage, while chlorine (20,000 ppm) or lactic acid (2.5%) treatment took longer time to reach the same level. With sprouts, CA-LA coating resulted in > 2.5 log reduction for pathogen cells. In contrast, log reduction was < 0.6 for either chlorine (20,000 ppm) or lactic acid (2.5%) treatment. In general, this study indicated the effect of calcium alginate coating on reducing bacterial load of alfalfa seeds and sprouts, however, the germination rate of treated seeds was compromised due to the addition of lactic acid in the seed coating. Further study is needed to select antimicrobial compounds with minimum impact on germination rate of seeds.</div><div><br></div>

In this study we investigate the synergistic effect of low frequency ultrasonication (14, 22, and 47 kHz) on antimicrobial activity of Cecropin P1 against Escherichia coli. The hypothesis was tested by comparing three different treatments (1) ultrasonication only (2) Cecropin P1 only (3) combination of both. The results showed that the combined treatment deactivate E. coli more efficiently by six order of magnitude. The mechanism of membrane permeabilization due to Cecropin P1 is also investigated using dye leakage experiment. The result indicated pore formation and carpet mechanism. Finally, a mathematical modeling is proposed to explain the synergistic effect, allowing us to make better prediction for cell deactivation.

<p>Cellulose nanocrystals (CNCs) and its composite coatings may impart many benefits in packaging, electronic, optical, etc. applications; however, large-scale coating production is a major engineering challenge. To fill this knowledge gap, a potential large-scale manufacturing technique, roll-to-roll reverse gravure processing, has been described in this work for the manufacture of CNC and CNC-poly(vinyl alcohol) (PVA) coatings on a flexible polymer substrate. Various processing parameters which control the coating structure and properties were examined. The most important parameters in controlling liquid transfers were gravure roll, gravure speed, substrate speed, and ink viscosity. After successful fabrication, coating adhesion was investigated with a crosshatch adhesion test. The surface roughness and morphology of the coating samples were characterized by atomic force microscopy and optical profilometer. The Hermans order parameter (S) and coating transparency were measured by UV–Vis spectroscopy. The effect of viscosity on CNC alignment was explained by the variation of shear rate, which was controlled by the micro-gravure rotation. Finally, the CNC alignment effect was investigated for gas barrier and thermal management applications.</p> <p>In packaging applications, cellulose nanomaterials may impart enhanced gas barrier performance due to their high crystallinity and polarity. In this work, low to superior gas barrier pristine nanocellulose films were produced using a shear-coating technique to obtain a range of anisotropic films. Induction of anisotropy in a nanocellulose film can control the overall free volume of the system which effectively controls the gas diffusion path and hence, controlled anisotropy results in tunable barrier properties. The highest anisotropy materials showed a maximum of 900-fold oxygen barrier improvement compared to the isotropic arrangement of nanocellulose film. The Bharadwaj model of nanocomposite permeability was modified for pure nanoparticles, and the CNC data were fitted with good agreement. Overall, the oxygen barrier performance of anisotropic nanocellulose films was 97 and 27 times better than traditional barrier materials such as biaxially oriented poly(ethylene terephthalate) (BoPET) and ethylene vinyl alcohol copolymer (EVOH), respectively, and thus could be utilized for oxygen-sensitive packaging applications. </p> The in-plane thermal conductivity of CNC - PVA composite films containing different PVA molecular weights, CNC loadings and varying order parameters (S) were investigated for potential application in thermal management of flexible electronics. Isotropic CNC - PVA bulk films with 10-50 wt% PVA solid loading showed significant improvement in thermal conductivity compared to either one component system (PVA or CNC). Furthermore, anisotropic composite films exhibited in-plane thermal conductivity as high as ~ 3.45 W m-1 K-1 in the chain direction, which is higher than most polymeric materials used as substrates for flexible electronics. Such an improvement can be attributed to the inclusion of PVA as well as to a high degree of CNC orientation. The theoretical model was used to study the effect of CNC arrangement (both isotropic and anisotropic configurations) and interfacial thermal resistance on the in-plane thermal conductivity of the CNC-PVA composite films. To demonstrate an application for flexible electronics, thermal images of a concentrated heat source on both neat PVA and CNC-PVA composite films were taken that showed the temperature of the resulting hot spot was lower for the composite films at the same power dissipation.

<p>  </p> <p>Fouling is a severe problem for food processing equipment, including heat exchanger, filtration system, etc., which can decrease heat transfer efficiency, increase pressure drop, and affect food quality and safety. To ensure process efficiency and product quality, regular cleaning is necessary. On manufacturing lines in the food industry, cleaning is usually performed by cleaning-in-place (CIP) operations, which mainly comprise water rinse, alkaline wash and acid wash steps. Although CIP can ensure uniform cleaning of equipment, lower costs associated with labor and plant downtime, and improve personnel safety, it consumes large amounts of energy, chemicals and water and thus affects the environmental sustainability.</p> <p>Microbubbles (MBs) are fine gas bubbles with very different physicochemical properties from millimeter-sized bubbles, including longer residence time in liquid, higher mass transfer rate, larger surface-area-to-volume ratio, and generation of free radical when bubbles collapse. In addition, MBs feature hydrophobic liquid-gas interface, allowing hydrophobic and amphipathic substances to attach to and spread on bubble surfaces. MBs have been used in cleaning processes, such as oil flotation and fresh produce washing; however, their applications in CIP operations in food processing have not been explored.</p> <p>The objective of this dissertation is to develop a novel CIP operation with the incorporation of MBs for cleaning of food processing equipment. MBs were incorporated into rinsing water to clean milk deposit fouled on heat transfer surface. A computational fluid dynamics model was built to predict the contact frequency of MBs with deposit and further identify the flow conditions that provided maximum MB-deposit contact. Moreover, MBs were confirmed to be able to attach to milk deposit by microscopic imaging. Rinsing with MB-incorporated water noticeably enhanced the deposit removal at Re of 4392 and 5403, by 27−31%. For cleaning of microfiltration membrane reversibly fouled by palm oil-in-water emulsions as model wastewater, although adding MBs into alkaline wash increased the membrane flux recovery by 235%, increasing the crossflow velocity of MB-incorporated liquid did not guarantee the enhancement in cleaning performance. Lastly, a MB-assisted full CIP process was tested on an ultrafiltration system used for milk concentration. MB-assisted CIP showed an increased cleaning efficiency with up to 72% higher flux recovery than conventional CIP, and alkaline wash with MBs added was the major step accounting for enhanced protein removal.</p> <p>Overall, this dissertation proves the effectiveness of MBs in cleaning of different types of food deposits, and provides groundwork knowledge of MB incorporation into CIP operations for different food processing equipment. The results are expected to guide the scale-up of MB-assisted CIP processes that can reduce the water and chemical usage in food manufacturing sectors, ultimately improving both economic and environmental sustainability of the food industry.</p>

<p>Multiple studies have demonstrated atmospheric cold plasma (ACP) as an effective non-thermal technology for microbial decontamination, surface modification, and functionality alteration in food processing and packaging. ACP constitutes charged particles, such as positive and negative ions, electrons, quanta of electromagnetic radiation, and excited and non-excited molecules, which corresponds to its predominant reactive properties. However, in many of these applications, the interactions between plasma and the components in food matrix are not well-understood. The <b>overall goals</b> of this dissertation were to 1) evaluate the interactions between high voltage atmospheric cold plasma (HVACP) and microbes in liquid and semi-solid food; 2) investigate plasma transfer into semi-solid foods and determine the relationship between microbial inactivation and plasma transfer; 3) explore the interactions between plasma and proteins. </p> <p>The first study explored the microbial (<i>Salmonella</i> <i>enterica</i> serovar Typhimurium, <i>S</i>. <i>enterica</i>) inactivation efficacy of HVACP. The physicochemical interactions between HVACP and biomolecules, including an enzyme (pectin methylesterase, PME), vitamin C and other components in orange juice (OJ) under different conditions was also evaluated. Both direct and indirect HVACP treatment of 25 mL OJ induced greater than a 5 log reduction in <i>S</i>. <i>enterica</i> following 30 s of treatment with air and MA65 gas with no storage. For 50 mL OJ, 120 s of direct HVACP treatment followed by 24 h storage achieved <i>S</i>. <i>enterica</i> reductions of 2.9 log in air and 4.7 log in MA65 gas. An indirect HVACP treatment of 120 s followed by 24 hours storage resulted in a 2.2 log reduction in air and a 3.8 log reduction in MA65. No significant (<i>P </i>< 0.05) Brix or pH change occurred following 120 s HVACP treatment. HVACP direct treatment reduced vitamin C content by 56% in air and PME activity by 74% in air and 82% in MA65. These results demonstrated that HVACP can significantly reduce <i>Salmonella</i> in OJ with minimal quality degradation.</p> <p>The second study in this dissertation examined the penetration process of plasma into semi-solid food and the resulting microbial inactivation efficacy. Agar gels of various densities (0.25, 0.5, 1.0, and 2%) with a pH indicator were inoculated with <i>S</i>. <i>enterica</i> (10⁷>CFU) and exposed directly (between the electrode) or indirectly (adjacent to the plasma field created between the two electrodes) to 90 kV at 60 Hz for up to 1.5 h. A long treatment time (1.5 h) caused sample temperature to increase 5~10 °C. The microbial analysis indicated a greater than 6 log₁₀ (CFU) reduction (both with air and MA65) in the zone with a pH change. Inactivation of bioluminescence cells in the plasma penetrated zone confirmed that the plasma, and its generated reactive species, inactivate microbial as it penetrates into the gel. A two-minute HVACP direct treatment with air at 90 kV induced greater than 5 log₁₀ (CFU)<i> S</i>. <i>enterica </i>reduction in applesauce. <em></em></p> <p>The third study investigated the interactions between HVACP and protein, using bovine serum albumin (BSA) as a model protein. The physicochemical and structural alteration of BSA and its reaction mechanism, when subjected to HVACP, were investigated. After treating 10 mL of BSA solution (50 mg/mL) at 90 kV for 20, 40, or 60 min, we characterized structural alteration and side-group modification. FTIR spectroscopy, Raman spectroscopy, and circular dichroism analysis indicated protein unfolding and decreased secondary structure (25 % loss of α-helix, 12% loss of β-sheet) in HVACP treated BSA. Average particle size in the protein solutions increased from 10 nm to 113 µm, with a broader distribution after 60 min HVACP treatment indicating protein aggregation. SDS-PAGE and mass spectrometer analysis observed a formation of new peptides of 1 to 10 kDa, indicating that the plasma triggered peptide bond cleavage. Chemical analysis and mass spectrometer results confirmed the plasma modifications on the side chains of amino acids. This study reveals that HVACP treatment may effectively introduce structural alteration, protein aggregation, peptide cleavage, and side-group modification to proteins in aqueous conditions, through several physicochemical interactions between plasma reactive species (reactive oxygen species and reactive nitrogen species) and the proteins. This finding can be readily applied to other plasma-protein studies or applications in the food system, such as enzyme inactivation or protein-based film modifications.</p>

<p>Zein is a major storage protein of corn, with unique amphiphilic film forming properties. It is insoluble in water, but soluble in 70% ethanol and acetic acid, and has been declared ‘generally recognized as safe’ (GRAS) by the FDA. Due to new advances in food nanotechnology, zein is being investigated for various applications such as biodegradable packaging, oral delivery of proteins and peptides, scaffold for tissue engineering, as well as biodegradable sensor platforms. The time consuming and highly complicated methods for toxin and allergen analysis in the food industry necessitates the need for a rapid, selective, compact and easy-to-use method of detection for analytes. In the scope of this dissertation, we investigated the feasibility of functional zein nanocomposite films and formation of a zein nanocomposite sensor assembly for rapid and highly selective electrochemical measurements of food toxins and allergens. Fabrication of a zein based electrochemical amperometric sensor assembly was studied, first through the comparison of various zein film characteristics changes with the application of Laponite®, graphene oxide and carbon nanotube nanoparticles, followed by a proof-of-concept study by detecting the gluten allergen protein gliadin. </p> <p>To mechanistically study the functional zein nanocomposite films, Laponite®, a silica nanoparticle, was added in the presence of 70% ethanol solvent and oleic acid plasticizer. The films were studied using various characterization techniques like transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), water contact angle measurements etc. Through Si-N bond formation between Laponite® and zein, fabricated zein nanocomposite films showed increase in surface hydrophobicity, water vapor barrier properties, tensile strength and Young’s modulus. Graphene oxide (GO), a carbon nanoparticle, was also incorporated into zein through the solvent casting process. Uniform dispersion of GO nanoparticles within zein matrix were confirmed up to 1% GO loading, and covalent and hydrogen bonding mechanisms were proposed. Similar to zein-Laponite® (Z-LAP) nanocomposites, zein-GO (Z-GO) showed increase in hydrophobic tendencies, rougher surface and a 300% improvement in Young’s modulus and 180% improvement in tensile strength at only 3% GO loading. Both nanoparticles increased tensile strength, thermal stability and water vapor barrier property of the films, indicating a potential for food packaging as an alternative application for the nanocomposite films.</p> Finally, the research focused on the fabrication of an electrochemical amperometric sensor, capable of detecting the protein gliadin, which is responsible for the allergic reaction with people having celiac disease. Novel biodegradable coatings made from zein nanocomposites: zein-graphene oxide, zein-Laponite® and zein-multiwalled carbon nanotubes (Z-CNT) using drop casting technique were tested for fabricating the electrochemical sensors using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) techniques. As Z-CNT produced the strongest signals compared to other nanomaterials, the active tip of the electrochemical sensor was functionalized through a sequence of layer by layer deposition of Z-CNT nanocomposite, antibody and target analyte. Here, Z-CNT acts as a natural linker molecule with large number of functional groups, that causes immobilization of capture antibody and target, to ensure high sensor performance. Both CV curves and SWV curves indicated successful sequential immobilization of gliadin antibody onto the Z-CNT coated electrode. The Z-CNT biosensor was successfully able to give CV signals for gliadin toxins for as low as 0.5 ppm and was highly specific for gliadin in the presence of other interfering molecules, and remained stable over a 30-day period. The low-cost, thin, conductive zein films offered a promising alternative for protein immobilization platforms used in sensors and can be extended to other matrices in biosensors as well as other functional film applications