Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agricultural and Environmental Sciences
Non Technical Summary
There has been a growing appreciation of the benefits of non-chemical-based treatments as a greener process for pathogen inactivation on food contact surfaces and in beverages. As a result, there is increased interest in applying ultraviolet light (UV) technology to inactivate bacterial biofilm-formers and on food contact surfaces the food industry. Major hurdles and challenges of using UV-C photons to decontaminate food contact surfaces include low light penetration through microbial biofilms. The researchwillbe furtherexpanded into beverages where novel optical devices will be evaluated. We hypothesize that effective inactivation of target microbial biofilm-forming pathogens on surfaces can be achieved through complete understanding of their spectral characteristics together with effective UV dose delivery. This project is intended to address the challenges of UV technologies by developing a novel high-power UV Light emitting diode (LED) system and by assessing the sensitivity of foodborne biofilm-forming bacterial pathogens and spores using the generated-germicidal UV regime. This proposal is targeted to develop decontamination strategies for foodborne biofilm-forming pathogens (Listeria monocytogenes, Salmonella enterica and Escherichia coli O157:H7) and on food-contact surfaces and beverages and account for their spectral characteristics. This project is aimed at engineering, design, and validation of UV LED systems that can deliver the efficient inactivation dose and to determine inactivation kinetics of the target bacteria and spores. The novel system developed using this approach in this project will result in a breakthrough in current practices of surface and fluid disinfection in the United States and is likely to extend to finding solutions for global food safety issues.
Animal Health Component
25%
Research Effort Categories
Basic
25%
Applied
25%
Developmental
50%
Goals / Objectives
Food safety is an important component of public health world-wide. Every year, more than 16% of the U.S. population acquire a foodborne illness, and 3,000 people are killed by consuming contaminated foods (http://www.cdc.gov/foodborneburden/index.html). Also, the United States is burdened by more than $50 billion in economic costs related to foodborne illnesses each year (Erickson et al., 2010). The contamination and persistence of pathogenic bacteria and viruses in foods have become an emerging concern. Ready-to-eat fruits and vegetables including juices/beverages may contain human pathogens among their microflora owing to contamination at some point in the process from cultivation to consumption (Montgomery and Banerjee, 2015). Human enteric pathogens including E. coli O157:H7, hepatitis A virus (HAV), and human norovirus (NoV) and other pathogens have each been implicated in outbreaks of food-borne illness associated with consumption of fresh produce items including juices (Ponka et al., 1999; Anderson et al., 2001; Centers for Disease Control and Prevention (CDC), 2003; Schmid et al., 2007; Grant et al., 2008).Cross-contamination in the food industry is defined as direct or indirect microbial transference from a contaminated to a non-contaminated matrix, which can be food, work surfaces, or workers, among others. In contrast, recontamination refers to the contamination of food after it has undergone a sanitizing treatment (Carrasco, Morales-Rueda, & Gar?ia-Gimeno, 2012). Cross-contamination plays a critical role in transmitting pathogens to food (Kusumaningrum, Riboldi, Hazeleger, & Beumer, 2003). Microorganisms colonize by adhering to living or inert surfaces, growing, and forming a self-produced polymeric matrix in which multiple microbial species may converge, known as biofilm (Carpentier & Cerf, 1993; Satpathy, Sen, Pattanaik, & Raut, 2016). These masses of cells further become large enough to entrap organic and inorganic debris, nutrients, and other microorganisms, leading to the formation of a microbial biofilm (Kumar & Anand, 1998). Mixed biofilms are reported to have higher resistance to disinfectants such as quaternary ammonium compounds (QAC) and other biocides. Since complex microbial communities in biofilms develop resistance to current physical and chemical disinfection methods2, there is a critical and urgent need to develop effective approaches to disinfect biofilms on food contact surfacesIn comparison to biological contamination; chemical contamination is another public heath risk. Among chemical contaminants, Aflatoxins are the most prominent one, produced by Aspergillus species (A. flavus, A. parasiticus, A. nomius, A. bombycis, A. ochraceoroseus, and A. pseudotamari). Majorly, aflatoxins are subdivided into B1, B2, G1 and G2 (Dhanasekaran et al., 2011). Climatic factors of tropical region favor the production of aflatoxins in commodities like nuts, spices, cereals, maize, soybean, peanuts, pistachios, cotton and rice (Ukwuru et al., 2017). Agricultural commodities, especially peanuts and peanut-based foods, are highly prone to aflatoxin contamination (Liu et al. 2010). Significant quantities of aflatoxins were observed in soybean, rice, corn, pasta, condiments, milk, dairy products and edible oil products (Richard, 2007; Dhanasekaran et al., 2011, FAO/WHO (2009, Binder et al, 2007). The ability of mycotoxins to remain stable throughout food processing is a concern (Bullerman and Bianchini, 2007). Among all the foods implicated in mycotoxin contamination, AFB1 is the most commonly mycotoxin primarily found in meat, milk and nuts. Aflatoxins are mutagenic, carcinogenic, teratogenic, hepatotoxic, and have immunosuppressive properties. Sufficient evidence on the carcinogenic potential of AFB1 led the International Agency for Research on Cancer (IARC) to categorize AFB1 under Group 1 carcinogens. AFM1 is considered to be possibly carcinogenic to humans and hence, IARC placed it under Group 2B carcinogens (IARC. (2012).Smart approaches and control strategies that prevent growth of pathogenic organisms and degrades mycotoxins, remain crucial for providing safer foods. Tennessee State University [TSU] propose a novel approach to decontaminate food contact surfaces [stainless steel, rubber, glass, plastic] and opaque beverages using High Intensity Ultraviolet Light Emitting Diodes [LED] technology. We propose an integrated project to investigate the effect of high intensity UV-C photons on bacterial biofilm-forming communities on surfaces. A secondary goal is to develop UV dose response curves of pathogens in opaque beverages using targeting wave-lengths. This study utilizes sophisticated computing algorithms in quantifying the optical properties [absorption coefficient, reduced scattering and scattering anisotropy] of the biofilm or opaque beverages; this will enable delivery of accurate and quantifiable UV dose on surfaces. The novelty of the current proposed work is improved penetration capability of UV light technology via [A] selection of appropriate germicidal wavelength (where light absorption of biofilm is low) and [B] development of UV dose response curves for biofilms and viruses on various food contact surfaces and in opaque beverages; account for spectra characteristics. In addition, this study will determine the frequency of UV light exposure with respect to age and thickness of complex biofilms for efficient decontamination of surfaces.Within this project the team will focus on the application of highly energetic photons at wave-length from 254- 300 nm (high selective/tunable) on the inactivation of viruses and bacterial spores including biofilms. An important aspect of the study is the creation of science-based knowledge and bridge existing knowledge gaps by assessing the sensitivity of target foodborne bacteria, viruses and bacterial biofilms using appropriate wave-lengths of exposure.Project HypothesesWe hypothesize that effective inactivation of target microbes in opaque beverages and biofilm-forming pathogens on surfaces can be achieved through complete understanding of their spectral characteristics together with effective UV dose delivery. This project is intended to address the challenges of UV technologies by developing a novel high-power UV Light emitting diode (LED) system and by assessing the sensitivity of foodborne bacteria and foodborne biofilm-forming bacterial pathogens using the generated-germicidal UV regime.Goals and ObjectivesThe project members will work closely to achieve 4 key objectives that are structured into 4 work packages (WP). The major goal is to demonstrate and validate UV LED technology's potential to disinfect pathogenic bacterial biofilms and viruses (surrogates) on food contact surfaces including opaque beverages.Perform effective management of all project work and resources;Evaluate UV light spectra of target microbial biofilms and opaque beverages, assess optical properties & quantify UV dose using artificial neural networks;Design and develop a novel high-power UV LED system and develop UV dose response curves for simple and complex biofilms (L. monocytogenes, E. coli O157:H7) with respect to biofilm thickness;Develop UV dose response curves of L. monocytogenes, S. enterica, E. coli O157:H7, Bacillus cereus in opaque beverages using selected germicidal wave-lengths (including action spectra);
Project Methods
Biofilm formation on surfaces: A CDC Biofilm Reactor (model CBR 90; BioSurface Technologies, Bozeman, MT) will be used to grow biofilms (mono & mixed). Coupons with a diameter of 1.27 cm and thickness of 0.3 cm, of different materials will be obtained from BioSurface Technologies: stainless steel type 304, rubber, borosilicate glass. First, coupons will be installed inside the CBR in triplicate and the CBR unit will be sterilized and then filled with 350 mL of Tryptic soy broth (TSB) or selective media. An overnight liquid culture (mono or mixed) of ~108 CFU/mL (L. monocytogenes, E. coli O157:H7) will be inoculated into the CBR at 1% of the CBR volume. The CBR will be operated in batch-phase for 24 h at 120 rpm with fresh TSB pumped continuously (flow rate 0.8 mL/min) for another 48 h at 25 °C. After each run, the CBR will be dismantled and coupons removed aseptically. Biofilms will be grown to addition 72 to 144 hrs. to get mature biofilms for further testing.Microbial Analysis (Bacterial strains and growth conditions): Three vegetative bacterial strains and one bacterial endospores strain will be used in this study: E. coli ATCC 25922, L. monocytogenes ATCC 19115 and B. cereus ATCC 14579. These cultures will be obtained from the American Type Culture Collection (ATCC). E. coli, L. monocytogenes will be grown by two successive transfers of individual strains incubated at 37 °C for 18 h in 50 mL Tryptic Soy Broth (TSB) (Oxoid Ltd.) or selective media. Then, all strains will be grown to 1 L level using 5 % (50 mL) inoculum. B. cereus ATCC 14579 will be cultured using Bacto Brain Heart Infusion broth (BHI, Beckton Dickinson, Franklin Lakes, NJ) as a growth medium, incubated at 37 °C with aeration at 180 rpm for 18 h. Thereafter, a nutrient-rich, chemically defined medium Mineral Salts Medium (MSM) described previously will be used to obtain spores (Garcia et al., 2010). Sporulation and spore handling will be performed as followed: 20 mL of an overnight-grown culture will be used to inoculate 200 mL of MSM media in 1 L flasks and incubated at optimal sporulation temperature of 30 °C with aeration at 180 rpm for 3 days.Optical Property Measurements: Optical property measurements procedure will be carried out as described by Jacques (2013, Patras et al., 2017; Patras et al., 2019; Gunter-ward et al., (2018).UV Dose/Fluence Determination: Batch scale irradiations will be performed using a UV LED "Collimated Beam" device. This apparatus was designed to provide uniform, quanti?ed irradiation to liquid samples, and the associated methods, including calibration, ?uence determination, and quality assurance protocols, have been developed and standardized in the ?eld of water disinfection (Bolton & Linden, 2003). To enhance mixing, irradiated volumes will be reduced to 5 mL samples in 10 mL beakers, with continuous stirring. All optical parameters and UV fluence calculations are described in our recent publication (Patras et al., 2017; Islam et al., 2016a, Islam et al., 2016b; Patras & Sasges, 2018; Chandra et al., 2017). Energy of photons can be calculated as shown below. UV irradiations using a bench scale UV LED system: Test fluids will be irradiated using a UV LED system. The reactor system will bedesigned to achieve good mixing and uniform fluence to the test fluid. A collimated tube will be connected to the LED bank to collimate photons, view angle of the photons will be adjusted to account for off axis photons . Coupons containing biofilms or beverages inoculated with bacteria (7-log CFU ml-1) will be placed aseptically at the center of the light emitting area and exposed to different UV doses.HPLC analysis of Mycotoxin Destruction: A Shimadzu HPLC system equipped with a florescence detector, (Model: RF20A) will be used for analysis of AFB1, AFM1. The method uses a 250-mm long Supelco® C18 column, 4.6 mm, 5µm (Phenomenex, California, USA) as a stationary phase and a mixture of water/acetonitrile/methanol in the ratio of 60:20:20 as the mobile phase. Aflatoxins (AFB1, AFM1) will be detected using emission and excitation wavelengths of 365 nm and 440 nm respectively. Separation will be achieved at room temperature under isocratic flow mode with a flow rate of 1 ml. min-1. The LOD and LOQ values will be calculated considering a signal-to-noise ratio of 3:1 and 5:1, respectively (Patras et al., 2017).Aflatoxins degradation analysis LC-MS/MS: The identification of AFB1, AFM1, and the respective degraded products will be carried out with an LC-MS method using a Shimadzu Prominence XR UHPLC system (Shimadzu Scientific Instruments, Columbia, MD) which included two Shimadzu LC-20ADXR pumps, a SIL-20ACXR autosampler, a CTO-20A column oven, and a Shimadzu LCMS 8030 triple quadrupole mass spectrometer. Chromatographic separation will be achieved with Phenomenex Kinetex 2.6 C18 column (50 × 2.1 mm, 2.5 μm) maintained at a temperature of 40 °C. The mobile phase will consist of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate will be 0.5 mL/min. Initially, solvent B concentration will be 5% and will increase linearly to 95% until 4.5 min. Solvent B will be returned to 5% at 4.51 min and will remain at 5% until the end of the time program at 6.0 min. The injection volume will be 5 μL. For LC-MS detection, an electrospray ionization source will be utilized in the positive ion mode with the following parameters: DL temperature, 250 °C; nebulizing gas flow, 3.00 L/min; heat block, 450 °C; and drying gas flow, 20 L/min. Control and experimental samples will be injected and data acquired in the scan mode to search for degraded products. For any potential degraded products, an LC-MS/MS product ion scan method was set up with to scan for products between 100 and 500 m/z at 15,000 amu per second with the appropriate precursor ion identified in scanning mode. Selected ion monitoring (SIM) MS methods will be developed for monitoring the following ions: AFB1 (m/z 313), AFB1-degraded products (m/z 303, m/z 311, and m/z 331), AFB1-degraded product (m/z 301), Patras et al., 2017).Cell viability assay (cytotoxicity assessment of irradiated beverages): HepG2, CCD-18Co and HCT-116 cells will be seeded in a 24-well plate at a seeding density of 2X105 cells per well. HepG2 and CCD-18Co cell will be grown in Eagle's minimum essential medium (EMEM) while HCT-116 will be grown in McCoy's 5a medium. All the cells will be supplemented with 10% fetal bovine serum (FBS) and incubated in a humidified chamber at 37 °C and 5% CO2 condition. Following 24 h, the cells will be serum starved overnight in a 1 mL respective media containing 1% FBS. The cells will be treated with 10 ml of the reconstituted samples for either 12 or 24 h. At the end of the exposure time the cell viability will be measured using CCK-8 reagent as per manufacturer's instruction (Dojindo Molecular Technologies, Inc., Rockville, MD). The absorbance was read at 450 nm with a reference wavelength of 650 nm in a Synergy 2 multi-mode microplate reader (BioTek, Winooski, VT). At the end of the sample exposure to the mammalian cells, the cell suspensions will be centrifuged at 400 g for 5 min and supernatant will be collected for the estimation of LDH and cytokines. For the determination of LDH, a 100 μl of supernatant will be added to 96-well plate containing 100 μl of reaction mixture (Roche Applied Sciences). The plate will be incubated for 30 min at room temperature. The reaction will be stopped using a stop solution and absorbance measure at 492 nm wave length with a reference wave length at 690 nm.