STEERING INNOVATION FOR TREATMENT OF LIQUID FOODS TO ELIMINATE PATHOGENIC MICROBES AND TOXINS USING LOW WAVE-LENGTH UV IRRADIATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1005352
• Grant No.: 2015-69003-23117
• Project No.: TENX-2014-06135
• Proposal No.: 2014-06135
• Program Code: A4131
• Project Start Date: Feb 15, 2015
• Project End Date: Feb 14, 2019
• Grant Year: 2015

Project Director
Patras, A.

Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209

Performing Department
Ag. & Environmental Science

Non Technical Summary
Ultraviolet (UV) treatment of water is a well-established technology, primarily applied for disinfecting drinking water and wastewater. UV penetrates relatively well into these fluids, enabling effective treatment of the bulk fluid. In recent years there has been a growing appreciation for the benefits of UV treatment, such as minimal byproduct formation compared with conventional chemical disinfection. As a result, there is a growing interest in applying UV technology for treating fluids in which UV does not penetrate easily (i.e. liquid food products, health drinks, sugar syrups, enzymatic solutions, cell culture media). These opaque fluids present new challenges for reactor modeling, design and performance testing. This project is intended to address these challenges by developing new techniques and concepts for reactor testing, modeling and design, extending conventional techniques into this new regime. Dose delivery protocols will be developed using the accurate optical properties which are the main parameters in dose calculations. Dose Delivery will be tested for a range of ultra-violet transmittance values (%/cm) and a range of viral surrogates of known UV sensitivity. The above described concepts and processes will be tested for a range of foods using a collimated beam bench system and a full scale-pilot reactor. Once established, UV irradiation at 254 nm will tested and assessed on a chemical contaminants, pathogens, allergens and bioactive molecules. The strengthened analytical and modeling capability and capacity will accelerate optimized product development, provide for better monitoring and process control, facilitate technology integration for enhanced or extended performance including risk reduction, and provide cost effective solutions to food industry. The rigors of computational and analytical approaches integrated with experimental approaches additionally provides market leading knowledge, and the knowledge base to address issues critical to validation of novel food process technologies being scrutinized by end users, consultants and regulators. The strategic objective of the research project is to operationally define and engrain through disciplined implementation, a holistic Roadmap for accelerating the innovation processes in food safety.

Animal Health Component

0%

Research Effort Categories

Basic

10%

Applied

40%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	5010	2020	100%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
5010 - Food;

Field Of Science
2020 - Engineering;

Keywords

bioassay

irradiation

liquid foods

microbes

safety

thin-film technology

uv technology

Goals / Objectives
Long-Term Goals And Project ObjectivesThe strategic objective of this research program is to operationally define and engrain within theconsortium through disciplined implementation, a holistic Roadmap for accelerating theinnovation process in irradiation research in partnership with AFC (Aquafine Corporation). The roadmap will guidetechnology development for contaminant treatment (microbe & toxins removal) by integrating,in a comprehensive framework, key aspects such as customer needs, techno-economic feasibility,process modeling, and technology developmentThe specific objectives of this project are:1. Perform effective management of all project work and resources;2. Study and assess the fluid optical attenuation coefficients [scattering, scatteringanisotropy, absorption and refractive index] at selected wave-lengthsProject Narrative23. Determine the UV dose delivery using optical parameters and microbial surrogates MS2(ssRNA virus) and T1UV (dsDNA) as dosimeters in a bench scale collimated beam, athin-film reactor system for a range of model and liquid foods;4. Set up, tune and optimize UV doses for a range of targets including toxins, bacteria,viruses and bioactive molecules, and estimate electrical energy consumption5. Perform chemical profiling and cytotoxicity analysis of UV irradiated liquid foods in cellculture models; and6. Integrate scientific research with student learning through knowledge transfer sessions.

Project Methods
Optical Property Measurements and SimulationOptical property measurements procedure will be carried out as described by Jacques (2013).UV Dose DeterminationUV dose determination will carried out by developing a MATLAB based Monte Carlo program to deduce the photon fluence in a 5 ml volume in a 10ml beaker. The simulations will automatically assume irradiance to be 1W/m2. This will be adjusted to get the fluence for the correct irradiance values. The program has been described by Wang (1995). Collimated Beam Set-UpAFC will assist in building and designing a collimated beam unit for irradiating samples (Figure 1). This unit will consist of a low-pressure lamp operating at 254 nm wave-length within built cooling device and an automatic timer. The CB beam will be designed and machined so as to deliver a uniform irradiance over a 5ml sample volume. A black plastic tube will be attached from top of the lamp to increase the degree of collimation. Bioassay & UV Sensitivity TestingIn the context of this document, an empirical assessment of the inactivation response of a specific microorganism to a controlled dose of UV light, usually in UV reactors/systems is termed as biodosimetry. In order to determine the sensitivity of the challenge organisms, irradiations will be performed in buffered water or test fluid using a collimated beam irradiation device. The test fluid will be normally placed on the horizontal surface below the bottom of the collimating tube. The intensity of the light will be controlled by the distance between the end of the collimating tube and the irradiation vessel. For clean water and microbial surrogates (target micro-organisms). The organism sensitivity is the inverse of the slope and expressed as mJcm-2. This parameter is very important in designing target doses for various microbial surrogates. The same procedure will be used for monitoring UV dose delivery in liquid foods. The dose delivery testing and UV sensitivity are based on the methods described in the UVDGM (USEPA, 2006).Chemical ProfilingAnalytical methods from the literature based on reverse- phase high performance liquid chromatography (diode array detector or evaporative light scattering detection) and gas chromatography-mass spectrometry for the determination of polyphenols, vitamins, sugars and amino acids will be adapted (Tsao & Yang, 2003; Ma et al. 2014; Neves & Morais, 1997). If possible the method will be streamlined in order to facilitate a high throughput of samples. The above listed compounds will be measured by reverse-phase high performance liquid chromatography (Agilent 1100 series and Shimadzu CLASS-VP System) equipped with a UV or ELSD detector. Separations will be performed on a classical C18 column using a binary solvent system. Limit of detection, limit of quantification and compounds recovery will be carried out to assess the validity of the methods. HMF will be quantified as per the method described by Arif?na et al. 2014), if present.Microbial Analysis (Bacterial strains and growth conditions) Salmonella enterica serovar Typhimurium ATCC 14028, Salmonella enterica serovar Montevideo ATCC 8387, and E. coli O157:H7 strains ATCC 700728, ATCC 35150, and Listeria monocytogenes strains ScottA and V7 (4b) will be used in this study. Strains of Salmonella and E. coli O157:H7 will be stored with 20% glycerol at −80 °C in tryptic soy broth, (Difco Co.) and L. monocytogenes in Listeria enrichment broth (Difco Co.).Growth and inactivation studies. A loop inocula obtained from frozen bacterial cultures will be respectively introduced to tubes containing 10 ml of tryptic soy broth or Listeria enrichment broth propagating medium and placed at respective incubating temperatures. The E. coli O157:H7 and Salmonella strains will be inoculated in tryptic soy broth tubes prior to incubation at 35 °C for 24 h. Listeria monocytogenes strains will be inoculated in Listeria enrichment broth (LEB) and incubated for 24 h at 37 °C. The E. coli O157:H7 and Salmonella cultures will be streaked on tryptic soy agar (TSA) plates and Listeria monocytogenes cultures will be streaked on Listeria enrichment agar plates.A single colony of each strain of E. coli O157:H7, Salmonella L. monocytogenes will be picked and then added to 50 mL tubes containing 25 mL of each selective medium and incubated overnight while shaken at 37 °C. 5 mL of the overnight of each strain of E. coli O157:H7, Salmonella L. monocytogenes will be transferred to 500 mL of fresh corresponding medium and incubated at 37 °C for 18 h to obtain early-stationary phase cells. Incubation for 24 h allowed the respective bacteria to approach the stationary phase of growth. For E. coli O157:H7, Salmonella, and L. monocytogenes strain inactivation trials, the overnight culture of each strain will be homogenized and distributed in ten 1 mL Eppendorf tubes that will be centrifuged at 1000 rpm for 2 min at an ambient temperature. 0.8 mL of the supernatant phase will be discarded and the other 0.2 mL of all ten tubes will be spread on 50 mL of fresh samples (liquid products), to obtain a concentration of cells around 8 log10 CFU/ml. Afterwards, 5 mL of the homogenized mixture will be transferred to a 10ml beaker for irradiation experiments. For enumeration, decimal dilutions will be made with 0.1% peptone water and samples will be surface plated in triplicate on respective medium. For Salmonella onto Xylose-Lysine-Desoxycholate (XLD) selective Agar (BD, Franklin Lakes, NJ) plates; E. coli O157:H7 onto sorbitol-MacConkey agar, supplemented with potassium tellurite and cefixime; and L monocytogenes will be plated on Listeria enrichment agar plates. The plates will be incubated at 37 °C for 24 h and counted. To determine the D10 value (mJcm-2) for different targets, log inactivation data will be plotted against selected doses.MS2 (single stranded RNA virus) and T1 (double-stranded DNA virus) enumeration and testing will be carried out at GAP ENVIROMICROBIAL SERVICES INC, Canada. The host used for T1 enumeration and propagation is E. coli CN13 ATCC 700609 and MS2 uses an E. coli host strain, ATCC 23631. GAP uses these two strains of E.Coli for virus culturing and platting. Cytotoxicity AnalysisThe complete cell culture media will be treated with a range of low, medium and high doses of UV. After irradiation, the media will be filtered through 0.2µm filters. Effects of irradiated culture media on human cells will be established using cell viability assay and colony formation assay. Normal human intestinal myofibroblasts cells (Lonza) will be maintained in non-irradiated culture media. Cells will be seeded in a 96-well plate. After 24 hours, cell culture media will be replaced with irradiated cell culture media. After another 24, 48, 72 or 96 hours of treatment, cells will be subject to MTT assay to determine cell viability as we previously established (Qiu et al., 2011). In colony formation assay, cells will be cultured in 6-well plates for 24 hours, and then treated with irradiated media for 10 days. The numbers and sizes of colonies formed will be quantified after staining with crystal violet (Nutakul et al., 2011). We will utilize flow-cytometry analysis for cell cycle and apoptosis to further determine the effects of irradiated media on the cell cycle progression and cell death of normal human intestinal myofibroblasts cells. As we previously described, cells will be cultured in 6-well plates for 24 hours, and then treated with irradiated media for 24, 48, 72 or 96 hours. Cells will be harvested, stained, and analyzed by flow-cytometry (Qiu et al., 2011).

Progress 02/15/15 to 02/14/19

Outputs
Target Audience:Target audiences reached by this integrated project includes equipment manufacturers and designers, regulatory agencies, food companies (Unique Food solutions, Vita Coco etc), faculty (research and extension) and graduate students. In particular one Beverage Company has shown considerable interest in UV technology and willing to invest in commericial aspects of the project. The team at TSU is still in talks regarding the Commercialization and planning extensive pilot test. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has supported the research training of 6graduate students (funded by college external funds), 2postdoctoral scholar, 4under-graduate (Deans scholar Program)and 4 high school students. Through this interdisciplinary training, these researchers were primarily engaged in the area of food engineering, food and analytical chemistry, basic and molecular microbiology. Interactions among the members have significantly generated cross-disciplinary collaborative research. The team members worked very closely with the industrial partners (Trojan Technologies and Aquafine Corporation (AFC)). The integration of industrial partners have strengthened TSU's capacity and opportunity to assist in commercializing novel UV irradiation processes and technologies for food safety applications. How have the results been disseminated to communities of interest?A workshop was conducted at Tennessee State University.This workshopaddressedimportant issues related to the validation of non-thermal processing methods used for controlling pathogens and mycotoxins in foods and ensuring compliance with regulatory requirements, such as those set by the Food Safety Modernization Act. Leading experts in Food Safety--including representatives of US regulatory agencies and of the food industry discussed best practices and challenges related to the validation and adoption of non-thermal technologies for microbial inactivation. This workshop also discussed critical issues in pathogenesis, modes by which different pathogenic bacteria causes illness to humans, virulence factors associated with pathogenic microbes, dissemination routes for pathogenic microbes and progression of infection. This symposium was made possible by a grant from the United States Department of Agriculture and contributions from the Department Agricultural and Environmental Sciences, College of Agriculture, Tennessee State University, and Industrial partners. It was attended by Tennessee State University and University of Tennessee extension agents. All results were shared with industrial stake holder group. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The main goal of this integrated project is to evaluate, improve and maximize the safety and quality (sensory) of UV processed foods and perform cytotoxicity analysis of UV irradiated fluids in cell culture systems. To achieve this goal, the team developed, standardized and validated a commercial pre-preproduction type UV system. Within this project, the team focused on the application of highly energetic photons at 254 nm on the inactivation of vegetative cells and model viruses in highly opaque liquid foods. During this reporting period, mathematical modelling of the process was conducted using a 2D-axial symmetric CFD approach. Navier Stokes and Radiative Transfer Equation equations were solved using Fluent Ansys software to predict the flow field and the fluence rate distribution using experimentally measured optical parameters (absorption and reduced scattering coefficient). Flow data were calculated for the thin-film reactor for clean water and test fluids at the entrance and exit sections of the device. Velocity (v = Q/Ainlet), Reynold's number (Re= (v x dinlet x p/(µ)) and theoretical residence time (Vreactor/Q) were calculated based on the measured flow rates. The residence time of the fluid was 10 seconds. The disinfection efficiency of the UV system will be higher when the turbulence of the flow is more intense. The thin film UV apparatus was developed and validated in collaboration with the industrial partner. It consisted of a three lamp thin-film annular reactor, consisting of inlet and outlet tanks, a peristaltic pump, a monochromatic low-pressure amalgam lamp emitting at irradiation at 254 nm wavelength. UV fluence delivered to the fluid was validated by biodosimetry using T1UV bacteriophage. Highly Opaque fluids were artificially prepared by adding concentrated humic acid to water until to get UV transmittance (%/cm) of 7.39E-12 ± 1.35E-12. pH of the fluid was adjusted to 7.1 ± 0.2. Test fluids were inoculated separately with test microorganisms and treated with UV-C light. After treatment, enumeration of spores or vegetative cells were performed by serial dilution plate count method. Populations of vegetative microorganisms were reduced by more than 5 log10 and bacterial endospores were reduced by 4.5 log10 at 600 Litre/hr (Re>2500, transitional flow). The inactivation kinetics of microorganisms were described by log linear models with low root mean square error (RMSE) and high coefficient of determination (R2 > 0.98) confirms similar dose distribution in each reactor. The D10 values were identified as 2.9 ± 0.1, 4.4 ± 0.3, 4.7 ± 0.2 and 9.3 ± 0.2 mJ⋅cm-2 for E. coli (ATCC 25922), S. enterica serovar Muenchen, L. monocytogenes (ATCC 19115) and B. cereus endospores (ATCC 14579) respectively. The relative RED bias for the system was around 1.05, indicating efficient mixing. The electrical energy per order (EEO) was less then 1kWh.m-3.log-1for E.coli, Salmonella and Listeria strains. In contrast higher EEO were calculated for bacterial endospores. This study clearly demonstrated that high levels of inactivation of microorganisms can be achieved in opaque fluids using UV-C light using thin-film reactor technology. This integrated projectadvancedthe feasibility of UV-C light technology to treat opaque liquid beverages. The next steps for this program to conduct more extensive pilot studies at the commericial sites. Currently TSU is partnering with Unique Food solutions and Trojan Technologies to conduct extensive pilot trials with thin-film technology. A workshop was conducted at Tennessee State University.This workshopaddressedimportant issues related to the validation of non-thermal processing methods used for controlling pathogens and mycotoxins in foods and ensuring compliance with regulatory requirements, such as those set by the Food Safety Modernization Act. Leading experts in Food Safety--including representatives of US regulatory agencies and of the food industry discussed best practices and challenges related to the validation and adoption of non-thermal technologies for microbial inactivation. The effect of non-thermal technologies on the nutritional quality and cytotoxicity was discussed. This workshop also discussed critical issues in pathogenesis, modes by which different pathogenic bacteria causes illness to humans, virulence factors associated with pathogenic microbes, dissemination routes for pathogenic microbes and progression of infection. This symposium was made possible by a grant from the United States Department of Agriculture and contributions from the Department Agricultural and Environmental Sciences, College of Agriculture, Tennessee State University, and Industrial partners.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Singh, Bhullar., Patras, A., Agnes Kilonzo-Nthenge., Pokharel, B.., Sasges, M. (2016). UV inactivation of bacteria and model viruses in coconut water using a collimated beam system. Food Science and Technology International (Accepted)
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Gopisetty, V., Patras A., Kilonzo-Nthenge, Pendyala, B., A., Sasges M. (2018). UV-C Irradiation as an alternative treatment technique: Study of its effect on microbial inactivation, cytotoxicity, and sensory properties in cranberry-flavored water. Innovative Food Science & Emerging Technologies 52, 66-74
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Advances and Implementation in Ultraviolet Light Technology in Beverage, Dairy, and Grain Applications Patras A. (2018). UV Treatment for Beverages: Considerations and Outcomes, presented at IFT, Chicago - 7/17/2018, 2:15 pm - 3:45 pm
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ward, D., Patras A., Pokharel, B., Sasges, M. Xiao H. (2018). UV-C irradiation on the safety of skim milk: effect on bacterial, viral inactivation and cytotoxicity. Journal of Food Process Engineering - DOI: 10.1111/jfpe.12944
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Pendyala, B., Patras, A. Gopisetty, V., Balamurugan, S., Sasges, M(2019). Evaluation of UV-C irradiation treatments on microbial safety, ascorbic acid and volatile aromatics content of watermelon beverage.
• Type: Book Chapters Status: Under Review Year Published: 2019 Citation: Patras, A., Pendyala, B., Bhullar M. (2019). Ultraviolet treatment of opaque liquid foods: From theory to practice. Elsevier Research works (Reference Module in Food Sciences)
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Pendyala, B., Patras, A. Gopisetty, V., Balamurugan, S., Sasges, M(2018)Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light. Pathogenesis and Disease Control (Accepted in press)

Progress 02/15/17 to 02/14/18

Outputs
Target Audience:Target audiences reached by this integrated project includes equipment manufacturers and designers, regulatory agencies, food companies,faculty (research and extension) and graduate students. In particular one Beverage Company has shown considerable interest in UV technology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has supported the research training of 5 graduate students, 1 postdoctoral scholar,two under-graduateand 4 high school students. Through this interdisciplinary training, these researchers were primarily engaged in the area of food engineering, food and analytical chemistry, basic and molecular microbiology. Interactions among the members have significantly generated cross-disciplinary collaborative research. The team members worked very closely with the industrial partners (Trojan Technologies and Aquafine Corporation (AFC)). The integration of industrial partners have strengthened TSU's capacity and opportunity to assist in commercializing novel UV irradiation processes and technologies for food safety applications. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Summary of the Proposed Work for Year 3/4 Aim 1: The team has develop a three UV reactor skid with assistance from Industrial partners (Aquafine Corporation). The main aim is to validate the system and conduct extensive CFD studies to quantify the RED dose for a range of flow-rates and varying UV sensitivies (D10). The team will develop UV dose response curves for a range of gram negative and gram postive microbes including viral surrogates.Once establised, a sizing table will developed. The sizing table will assist in developing applications for food companies.Aim 2: Preliminary data for EEO was generated. EEO is calculated to evaluate the amount of energy (kilowatt-hour) essential to for one log reduction of the concentration of a target bacterium (Bolton, 2001). Electrical energy per order (EEO) values for Escherichia coli O157:H7 ATCC 700728 and Salmonella Muenchen ATCC BAA 1764 are mentioned in Table 6. The EEO can largely differ from one system to another due to differences in design, dimensions and D10 value of the microbe being inactivated. A lower EEO value signifies lower energy consumption (Rodriguez, Buckow, Koutchma, & Balasubramaniam, 2015). Since the obtained D10 value of Salmonella Muenchen (3.14 mJ.cm-2) using continuous flow-through system is relatively higher than Escherichia coli (2.11 mJ.cm-2), the calculated EEO was higher for Salmonella Muenchen. EEO will calculated for a range of microbes including viral surrogates. Aim 3. Teaching and Outreach Activities: This activity represents development of educational programs at TSU as well as industry knowledge transfer sessions at Trojan Technologies and Aquafine Corporation. A new course in Food Processing has been recently developed (AGSC 5520) and section on optical technologies (UV irradiation) will be included in the graduate program in the Department of Agricultural and Environmental Sciences. A module on processes that improve the safety of food products, including UV irradiation technologies will be included in that course. It will include observations and labs in Dr. Patras laboratory. We plan to further build on this program with an overall aim to develop activities for the school curriculum in high schools. A workshop will planned in the summer of 2018 on UV technology. At this first Food Safety and Technology workshop hosted at Tennessee State University, we will address the importance of food safety throughout the food supply chain and communities that make up the ecosystem are all linked. Innovative technologies using non-thermal methods for food processing are of huge importance globally and consumer perception of these new technologies is critical for its implementation. With research case studies, educational based sessions, peer-to-peer conversations, and new technologies, we will provide a wide range of applicable, real solutions to identified needs and situations for the food community to address important food safety issues.The audience will learn about: Transmission of bacterial and viral pathogens and their pathogenesis; Novel non-thermal techniques for controlling pathogens (bacteria and viruses); Predictive Microbiology for Food Safety E-sensing techniques in food safety and quality analysis Consumer concerns and perspectives of newer food safety technologies

Impacts
What was accomplished under these goals? The overall research goal of this project is to evaluate, improve and maximize the safety and quality (sensory) of UV processed foods and perform cytotoxicity analysis of UV irradiated fluids in cell culture systems. To achieve this goal, the team developed, standardized and validated a collimated beam and a novel continuous flow UV reactor. The project team recently developed, validated and improved a flow-through UV system (bench) capable of delivering a tight UV dose. Using this reactor, the project team conducted a range of experiments and developed UV dose response curves for pathogenic and outbreak strains including bacterial spores. Case study 1 (milk treatment). The persistence of foodborne pathogens in milk has become a perilous public health concern as food borne outbreaks consistently occur as a result of pathogenic contamination (CDC, 2017). The efficacy of UV-C irradiation as a non-thermal processing method for skim milk was investigated. Milk inoculated with two surrogate viruses (MS2 and T1UV), and three bacteria (Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) was treated with UV-C irradiation. Biodosimetry techniques were used to calculate the reduction equivalent fluence (REF). Milk was irradiated using a Dean-flow spiral reactor with the fluid pumped around a central low-pressure mercury UV lamp (40 W) emitting at 254 nm wave-length. A series of known UV doses (0 - 168.33 mJ⋅cm-2) were delivered to the samples. The microbial loads of MS2, T1UV, Escherichia coli, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes were reduced by more than 5 log10. At the highest dose of 168.33mJ⋅cm-2, the results showed that UV-C irradiation effectively inactivated surrogate viruses in skim milk. This scientific study provided evidence based data on the advantages of UV-C light in microbial reduction at commercially applicable UV fluence in skim milk. The irradiated skim milk did not display any toxicity on mice liver and intestinal cells. UV-C irradiation is an efficient food preservation technology and offers opportunities for dairy and food processing industries to meet the growing demand from consumers for safer foods. This exploration would provide methodological evidence for commercialization of UV-C processing of milk and dairy based beverages. Case study 2 (Cranberry flavored water, CFW)). UV technology for treating acidic juices or beverages have recently begun to be commercialized. FDA approval in 2000 of UV irradiation to treat juice to reduce human pathogens in (21 CFR 179.39) requires that the UV irradiation be provided by low pressure mercury lamps emitting 90% of the emission at a wavelength of 253.7 nm (2537 A?), and that during the treatment, the juice undergo turbulent flow through tubes with a minimum Reynolds number of 2200 (FDA, 2000). To maintain quality and implement FDA guidance for acid beverages as mentioned above, acidic beverage producers can control pathogenic and spoilage microorganism by using UV-C light. The impact of UV-C irradiation at 254 nm on microbial inactivation, cytotoxicity, and sensory evaluation of cranberry flavored water (CFW) was studied. Two major pathogenic outbreak strains i.e., Escherichia coli ATCC 700728 and Salmonella enterica serovar Muenchen ATCC BAA 1764 were inactivated by more than 5 log10 at an UV-C fluence of 12 mJ?cm-2 and 16 mJ?cm-2 with D10 values of 2.11 and 3.14 mJ?cm-2 respectively. Cytotoxicity evaluation of CFW in human endothelial cells (EA.hy926) observed no toxic effects up to a maximum delivered UV-C fluence of 120 mJ?cm-2. Quantitative Descriptive Analysis (QDA) was performed to evaluate the sensory attributes of CFW at two (30 and 120 mJ?cm-2) UV-C fluence levels. Results suggested that UV-C irradiated CFW had no significant difference (p > 0.05) in the sensory attributes up to a fluence of 30 mJ?cm-2. Overall, this research suggests that UV-C treatment of CFW can achieve efficient microbial inactivation, without the generation of cytotoxic effects, and also can retain its sensory attributes. Cytotoxicity studies on irradiated human endothelial (EA.hy926) cells was conducted at fluence levels of 0, 15, 30, 60, and 120 mJ·cm-2. The results demonstrated that none of the UV-C fluence levels (0, 15, 30, 60, and 120 mJ·cm-2) caused cell inhibition viability in comparison with the untreated CFW. Case study 3 (coconut water). The production of low acid beverages such as coconut water must include dedicated bacterial spore inactivation steps to minimize the risk of spoilage or illness. Inactivation of bacterial endospores is a major concern in food industry especially for the processing of low acid (pH > 4.6) beverages. It is well known that spores can survive at these environments, and their subsequent vegetative growth when exposed to favorable conditions can lead to severe foodborne diseases and food spoilage. Clostridium botulinum (C. botulinum) is a group of four physiologically and phylogenetically distinct clostridia that share the common feature of producing the extremely potent botulinum neurotoxin (Lund and Peck 2000) which is a major concern in low acid beverages. Bacterial spores are generally more resistant than vegetative bacteria to ultraviolet inactivation. The UV sensitivity of these spores must be known for implementing UV disinfection. UV inactivation kinetics of bacterial spores in coconut water (CW) and sterile water (SW) was studied. Populations of Bacillus cereus and Clostridium sporogenes were reduced by more than 5.5 log10. At the highest dose of 90.5 mJ⋅cm-2, UV-C irradiation effectively inactivated Clostridium sporogenes in CW and SW by 6 log10. The dose required for one log inactivation (D10) was found to be 14.11 mJ⋅cm-2. For Bacillus cereus, D10 value was found to be 9.16 mJ⋅cm-2. No significant difference was observed in D10 values of spores suspended in the two fluid types (P>0.05). Energy absorbed for each UV dose (delivered) for test fluids was calculated. Comparative data between B. cereus and C. sporogenes for coconut water shows, absorbed energy varied significantly. Absorbed energy for B. cereus ranged from 180 to 902 J.L-1 for and 1141 to 3423 J.L-1 for C. sporogenes. This parameter considers the attenuation of UV energy in the UV reactor vessel and the fluid due to the reflection, refraction, absorption, and divergence (average energy distribution). The energy absorbed provides accurate information on the actual energy used for the microbial disinfection. Case study 4 (correlating microbial inactivation to DNA damage, milk as a model). UV irradiation disinfection is achieved by eliciting the formation of dimers which inhibits DNA replication as a result of the photochemical reaction. This study aimed to validate UV-C irradiation inflicted DNA damage in pathogens and assessed the cytotoxicity of irradiated milk. Milk inoculated with Escherichia coli, Salmonella Typhimurium, and Listeria monocytogenes was treated with UV-C irradiation followed by DNA damage assessment using conventional and next generation sequencing approach. Contaminated milk samples were irradiated using a collimated beam (CB) small batch reactor utilizing a low-pressure mercury UV lamp (40 W) emitting at 254 nm wave-length. Neutral Comet Assay was performed to assess the percent of DNA damage. The range of DNA damage for all bacteria was 39% to 72%. Currently, scale up equipment has already been developed by the research team and its efficacy in inactivating microorganisms and other spores in various beverages on a larger scale will be subject to future investigation.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Patras, A., Sasges, M. (2018). Corrigendum to UV dose measurement. https://doi.org/10.1016/j.foodcont.2018.02.015
• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Bhullar, M., Patras., A, Kilanzo-Nthenge A. Sasges, M. (2018). UV inactivation of bacteria and model viruses in coconut water using a collimated beam system. Food Science and Technology International,(Accepted with minor revisions).
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Gopisetty, V., Patras, A,Kilanzo-Nthenge A., Sasges, M., Xiao, H. (2018). Impact of UV-C irradiation on the quality, safety and cytotoxicity of cranberry flavored water using a novel continuous flow UV system. LWT- Food Science and Technology, https://doi.org/10.1016/j.lwt.2018.04.042
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Vergne, M.J., Patras, A., Singh, M., Pokharel, B., Shade, L., Xiao, H. & Sasges. (2018). UV-C Irradiation on the Quality of Green Tea: Effect on Catechins, Antioxidant Activity, and Cytotoxicity. Journal of Food Science (DOI:10.1111/1750-3841.14131)
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bhullar, M., Patras., A, Kilanzo-Nthenge A. Sasges, M. (2018)Microbial inactivation and cytotoxicity evaluation of UV irradiated coconut water in a novel continuous flow spiral reactor. Food Research International 103, 59-67
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Patras, A., Chandra, S., Bansode, R. R., Vergne, M.J. (2017). Effect of UV irradiation on aflatoxins reduction: a cytotoxicity evaluation study using Human Hepatoma cell line. Mycotoxins Research, DOI: 10.1007/s12550-017-0291-0
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Ward, D., Patras A., Pokharel, B., Sasges, M. Xiao H. (2018). UV-C irradiation on the safety of skim milk: effect on bacterial, viral inactivation and cytotoxicity. Food Control (FOODCONT-D-18-00410), submitted
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Gopisetty, V., Patras, A,Kilanzo-Nthenge A., Sasges, M., Si, H. (2018)UV-C Irradiation as an alternative treatment technique: Study of its effect on microbial inactivation, cytotoxicity, and sensory properties in cranberry-flavored water. FOODCONT-D-18-01182
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Pendyala, B., Patras, A. (2018). Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light. Food Microbiology (FM_2018_372)
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Ward, D., Patras A., Pokharel, B., Sasges, M. (2017). Efficacy of Ultraviolet (UV-C) Light in Reducing Foodborne Pathogens and Model Viruses in Milk. Journal of Food Processing and Preservation. DOI: 10.1111/jfpe.12586

Progress 02/15/16 to 02/14/17

Outputs
Target Audience:The target audiences reached by this project includes UV equipment manufacturers, regulatory agencies, food companies, academic faculty and researchers and graduate students. In particular one Beverage Company has shown considerable interest in UV technology. We are currently planning industrial trials at their manufacturing facility. The project team recently developed and validated a flow-through UV system capable in delivering a tight UV dose. Using this reactor, the project team is running trials to assess if UV (254 nm wave-length) can be used to inactivate spores (Clostridium Sporogenes) in low acid beverage products. Our preliminary data showed Clostridium Sporogenes has a D10 value of 20 mJ/cm^2 in buffer (pH 7). In contrast, a D10 value of 13 mJ/cm^2 was observed in test fluid (coconut water, pH 5). Based on this data, 12 log reduction can be achieved in a low acid beverage at a UV dose of 160 mJ/cm^2. The client has requested the team to conduct more inactivation trials. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research project has supported the research training of 5 graduate students, 1 postdoctoral scholar and two under-graduate students. Through this interdisciplinary training, these researchers were primarily engaged in the area of food engineering, food and analytical chemistry, basic and molecular microbiology. Interactions among the members have significantly generated cross-disciplinary collaborative research. The team members worked very closely with the industrial partners (Trojan Technologies and Aquafine Corporation (AFC)). The integration of industrial partners have strengthened TSU's capacity and opportunity to assist in commercializing novel UV irradiation processes and technologies for food safety applications. One of the distinguishing features of AFC's strategy for getting products to market has been the rigorous analysis and testing that products undergo during the design, development and validation phases. The graduate students and post doc fellow were trained in these strategies. Three Master's students graduated in year 2016. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Summary of the Proposed Work for Year 3 Aim 1: TSU team will develop a method to quantify the DNA damage and correlate it to inactivation (beverages studied in year 1 &2). The team is currently developing comet assay to assess DNA damage in irradiated microbes. In parallel, the team proposes to study the respiration rates of irradiated microbes and correlate in to microbial inactivation. Growth and respiration are the two fundamental and complementary life processes of metabolism in bacteria. Bacterial respiration can be estimated by measuring oxygen consumption or CO2 production rates, and likewise important as for the assessment of bacterial production. This will be measured using Li-Cor 6400 portable CO2 infrared gas analyzer. The team will attempt to correlate respiration rates with ATP production rates. Key Deliverables: Quantitative information on stress response of irradiated microbe. Aim 2: Based on the progress in years 1 and 2, the research goals for Year 3 are: Develop a sizing table (flow rate vs Reduction Equivalent Dose) for a bench scale continuous flow-through UV system. This information will aid in studying bacterial and viral inactivation in highly scattering fluids. A simple analogy is that the UV Dose is the amount of photons absorbed per surface area by an irradiated object during a particular exposure time. While UV dose delivered by UV system is often expressed as the product of the average UV intensity within the UV system and the theoretical treatment time, the experimental set-up gives intensity gradients within UV systems and gives rise to a distribution of delivered doses as opposed to a fixed value. Without proper mixing, fluid further from the lamp will receive a lower dose than that closer to the lamp. Typical UV irradiation research studies utilize batch reactors (i.e., collimated beam devices); however, continuous-flow reactors are significantly more desirable for industrial processes. Key Deliverables: Quantitative information on microbial, viral and enzyme inactivation using a continuous flow-through UV system. Aim 3: The overall goals of the Aim 3 in year 3 are: The team has develop a three UV reactor skid with assistance from Industrial partners (Aquafine Corporation). The members will develop a sizing table (flow rate vs Reduction Equivalent Dose) for continuous flow-through UV system (industrial type flow rates). A numerical analysis of a thin film reactor will be conducted to gain insight into dose distribution in opaque fluids (highly scattering or highly absorbing). The flow distribution in the fluid region will be simulated using ANSYS Fluent and the Radiative Transfer Equation will be solved using the Discrete Ordinates model, including both absorption and scattering. All modeling was conducted using a 2D axial-symmetric model. The CFD modelling and bioassay results will be compared. The team would optimize the validate a pilot reactor and study microbial, viral and enzyme inactivation. Mixingcapability of the reactors will also be studied. All tests will be performed at high flows to achieve a Reynolds number (Re) of approximately 3500 (turbulent flow). Deliverables: Quantitative information on microbial, viral and enzyme inactivation using an industrial type UV system ( 83 Watts/reactor, flow- rates: 10- 18 L/min). Aim 4: Based on the findings of Year 1 and 2, research goals for Year 3 are: Continue optimization of the industrial UV systems for beverage treatment (highly absorbing and scattering). Conduct extensive experimentation in order to calculate the electrical energy efficiency. The most common parameter in electrical energy efficiency of UV reactors is the Electrical Energy per Order (EEO), which will be evaluated in the study and defined as the number of kilowatt hours of electrical energy required to reduce the concentration of a contaminant or bacteria by one order of magnitude in one m3 of fluid. This will be calculated for a range of bacteria and viruses. Key Deliverables: Quantitative information on electrical energy requirement of the UV system for disinfection. Teaching and Outreach Activities: This activity represents development of educational programs at TSU as well as industry knowledge transfer sessions at Trojan Technologies and Aquafine Corporation. A new course in Food Processing has been recently developed (AGSC 3690) and section on optical technologies (UV irradiation) will be included in the undergraduate in the Department of Agricultural and Environmental Sciences. A module on processes that improve the safety of food products, including UV irradiation technologies will be included in that course. It will include observations and labs in Dr. Patras laboratory. We plan to further build on this program with an overall aim to develop activities for the school curriculum in high schools. This will fulfill USDA Research, Education and Economics Action Plan 2014 Strategic Goal #6 related to recruiting, cultivating, and developing the "next generation of scientists and leaders, and to produce a highly-skilled workforce for food, agriculture, natural resources and environmental sciences."

Impacts
What was accomplished under these goals? The overall research goal of this project is to evaluate, improve and maximize the safety of UV processed foods and perform cytotoxicity analysis of UV irradiated fluids in cell culture systems. To achieve this goal, the team developed, standardized and validated a collimated beam and a novel continuous flow UV reactor. When properly engineered continuous flow UV reactor have considerable potential to be a cost-effective solution for opaque fluid treatment disinfection. Given the high number of potential applications and the extreme diversity of opaque fluids (a consequence of their source and compositions), technical investigations were supported by market research in order to identify the market segment with the highest potential and establish business collaborations with early adopters. With this overall goal the key accomplishments of this project were: 1. Understanding microbial inactivation in highly scattering liquid foods (i.e milk). UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three pathogenic bacteria Escherichia coli ATCC 25922, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in buffer and milk were investigated. UV-C irradiation was applied to stirred samples, using a collimated beam operating at 253.7 nm wavelength. A series of known UV-C doses (0 - 40 mJ/cm^2) were delivered to the samples except MS2 where higher doses (0 - 150 mJ/cm^2) were delivered. At the highest dose of 40 mJ/cm^2, the three pathogenic organisms were inactivated by more than 5 log10. In order to calculate the UV dose in a given system, the optical properties of the fluid must be correctly determined. In this case study, the optics (absorption and scattering coefficients) of the fluid are accounted for, and dose delivery was verified through bio-dosimetry, ensuring that target levels of disinfection are achieved, and allowing direct comparisons with other UV-C treatment studies. This study clearly demonstrated that high levels of inactivation of pathogens can be achieved in milk, and suggests significant potential for UV-C treatment of treating fluids that exhibit significant scattering. 2(a). Study the effect of UV irradiation on microbial inactivation in coconut water. (2b) Study the effect of UV irradiation on nutritional quality and cytotoxicity of and green tea ((highly absorbing liquid food) 2a. This study investigated the ability of UV-C irradiation to inactivate microorganisms including bacteriophage in coconut water, a highly opaque liquid food. UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three pathogenic bacteria (E. coli ATCC 25922, Salmonella typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in peptone and coconut water were investigated. UV-C irradiation was applied to stirred samples, using a collimated beam operating at 253.7 nm wavelength. D10 values of all microbes were experimentally evaluated in buffer and ranged from (2.82 to 20 mJ/cm^2). Based in this information, a series of known UV-C doses (0 - 40 mJ/cm^2) were delivered to the samples except MS2 where higher doses were delivered. Inactivation levels of all organisms were proportional to UV-C dose. At the highest dose of 40 mJ/cm^2, three pathogenic organisms were inactivated by more than 5 log10 (p<0.05). It was also observed that L. monocytogenes and E. coli inactivation showed tailing. The same experiment was conducted using a continuous flow UV reactor. A series of known UV-C doses were delivered to the samples. Inactivation levels of all organisms were linearly proportional to UV-C dose (R2 >0.97). At the highest dose of 30mJ.cm-2, the three pathogenic organisms were inactivated by more than 5 log10. Results clearly demonstrated that UV-C irradiation effectively inactivated bacteriophage and pathogenic microbes in coconut water. It is quite evident that inactivation kinetics for all microbes followed first order kinetics values unlike previous studies with collimated beam approach which have reported concavity and pronounced tailing at higher UV doses. This may be attributed to the fact that the continuous reactor used in the present study induces adequate mixing in the fluid such that each fluid element received the same UV so as to provide uniform exposure. It is critically importance to make sure that a novel non-thermal technique such as UV irradiation does not produce toxic chemical compounds in the coconut water. Herein, we utilized human cells and mice liver cells to determine if UV-C irradiation affected the toxicity of coconut water. Our results showed that over the entire dilution range, untreated coconut water extract did not cause significant inhibition on the viability of human normal intestinal CCD-18Co cells and mice liver cells (data not shown). Results showed the effects of extracts of coconut water irradiated with different UV doses (100, 200, 300 and 400 mJ/cm^2) at 6.25 fold dilution on the viability of CCD-18Co cells and healthy mice liver cells. None of UV dosage caused increased inhibition on the viability of the cells in comparison with the untreated coconut water. These results suggest that UV irradiation at 100 to 400 mJ/cm^2 did not lead to the production of compounds cytotoxic to CCD-18Co cells and mice liver cells. UV-C irradiation did not cause production of cytotoxic compounds against human normal intestinal and mice liver cells in the coconut water. UV-C treatment did not change the cellular responses of both cell types to the coconut water extract. 2b. In this study, using a novel continuous flow UV reactor, the effect UV-C irradiation on the polyphenols of green tea was investigated. In addition, this study also evaluated the cytotoxicity of UV-C irradiated green tea on the colon cancer cell line (HCT-116) and fibroblasts from normal colon cells (CCD-18Co). UV-C irradiation doses ranging from 0 to 240 mJ/cm^2 were delivered to green tea, and polyphenols were chemically profiled. The results demonstrated that UV-C irradiation of green tea at relevant commercial disinfection doses induced minor reductions (p>0.05) in the concentrations of catechins. The irradiated green tea drink was evaluated for cytotoxic effects and showed no cytotoxic effects on normal intestinal cells, and both irradiated and non-irradiated samples exhibited very similar significant inhibitory effects on the growth of human colon cancer cells. The effects of UV-C irradiation of green tea was tested to determine if any changes on in vitro anticancer effects occurred as a result of UV-C exposure. The results showed that both UV irradiated and non-irradiated samples demonstrate green tea concentration-dependent effect to inhibit growth of human colon cancer HCT116 cells. Non-UV irradiated juice extract at 50-fold dilution caused significant inhibition on the growth of HCT116 in comparison to non-treatment control (p<0.05). In addition, all irradiated samples showed significantly higher inhibition as compared to non-irradiated controls. These results suggested that UV-C treatment at the doses of 17 to 240 mJ/cm^2 could slightly enhance efficacy of anticancer effects of green tea. Overall, these results demonstrate the effectiveness of the UV-C technology for treating highly turbid liquid foods such as green tea.2c. Overall, these accomplishments highlight the success of interdisciplinary research and its impact in developing novel non-thermal technologies to address food safety issues. These results suggest that UV-C treatment did not generate of cytotoxic compounds in coconut waterand green tea. Scale-up of the UV-C device, spore inactivation studies, and sensory changes in the beverage will be subject of further investigations. Scale up equipment has already been developed by the research team and its efficacy in inactivating microorganisms and other spores in various beverages on a larger scale will be subject to future investigation based on industrial partner feedback.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Vergne, M.J., Patras, A., Singh, M., Pokharel, B., Shade, L., Xiao, H. & Sasges, M. (2017). UV-C irradiation on the quality of green tea: effect on catechins, antioxidant activity, and cytotoxicity. Journal of the Science of Food and Agriculture (under review) JSFA-17-0413
• Type: Journal Articles Status: Other Year Published: 2016 Citation: Patras, A., Chandra, S., Bansode, R. (2016). Effect of UV irradiation on aflatoxins reduction: a cytotoxicity evaluation study using Human Hepatoma cell line. Mycotoxin Research (MYRE-D-17-00011); Accepted major revisions
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Singh, Bhullar., Patras, A., Agnes Kilonzo-Nthenge., Pokharel, B.., Sasges, M. (2016). UV-C inactivation of bacteria and model viruses in naturally opaque coconut water: A kinetic study.(Submitted, Food Biosciences)
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Singh, Bhullar., Patras, A., Agnes Kilonzo-Nthenge., Pokharel, B.., Sasges, M. (2016).Microbial inactivation and cytotoxicity evaluation of UV irradiated coconut water in a novel continuous flow reactor. Innovative food Science and emerging technologies. (Submitted, IFSET_2017_344)
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Islam, M.S., Patras, A., Pokharel, B., Wu, Y., Vergne, M.J., Shade, L., Xiao, H. & Sasges, M. (2016). Effect of UV irradiation on nutritional quality and cytotoxicity of apple juice. Journal of Agricultural and Food Chemistry Journal of Agricultural and Food Chemistry 64 (41), 7812-7822
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Islam, M.S., Patras, A., Pokharel, B., Wu, Y., Vergne, M.J., Shade, L., Xiao, H. & Sasges, M. (2016). UV-C irradiation as an alternative disinfection technique: Study of its effect on polyphenols and antioxidant activity of apple juice. Innovative Food Science & Emerging Technologies 34, 344-351
• Type: Journal Articles Status: Other Year Published: 2016 Citation: Chandra, S., Patras, A., Pokharel, B., Bansode, R. R., Begum, A., Sasges, M. (2017). Patulin degradation and cytotoxicity evaluation of UV irradiated apple juice using human peripheral mononuclear cells. Journal of Food Process Engineering (Accepted with minor revisions)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Co-authored publication (Patras et al), titled Effect of Ultraviolet-C irradiation and photosensitizers on mitigation of aflatoxins. IFT Annual Meeting + Food Expo, Chicago, July 16-19th , 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: UV-C Inactivation of Bacteria and Viruses in Coconut Water. International Association of Food Protection, St. Louis, Missouri July 31st  August 3rd, 2016
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Yannam, S; Patras, A; Sasges, M. (2016). UV-C irradiation for the inactivation of spoilage enzymes and its effect on physicochemical properties of coconut water. Journal of the Science of Food and Agriculture (JFSA-17-0024)

Progress 02/15/15 to 02/14/16

Outputs
Target Audience:As part of the project and our technolology adoption initiative, a beverage company has shown considerable interest in UV technology. We are currently planning industrial trials at their manufacturing facility.The project teamrecently developed and validateda collimated beam reactor for opaque fluid disinfection. Using this reactor, the project team is runningtrials to assess if UV (254nm wave-length) can be used to inactivate spores in low acid beverage products. Another objective is to verify the UV dose delivery in the fluids. Revently we showed that doses upto 120 mJ/cm^2 could be delivered to their products. The client has requested to verify UV doses upto 300 mJ/cm^2. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?3 Master's students (funded by Department of Agricultural and Environmental Sciences, Tennessee State University) and 1 Ph.D student (USDA funded)were hired in this project and the students were trained in optical measurements, dose delivery anddegradation kinetics. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Database of UV sensitivity of targets (D10) A database of UV sensitivities will be built in-house for a range of targets (Viruses: MS2, T1), (Bacterium: Salmonella enterica serovar Typhimurium ATCC 14028, Salmonella enterica serovar Montevideo ATCC 8387, and E. coli O157:H7 strains ATCC 700728, ATCC 35150, and Listeria monocytogenes strains ScottA and V7 (4b)); Toxins (Patulin, Ochratoxin A) using CB system. This data is required to design target doses in a batch or a continuous flow-through reactor. The targets has selected as per industrial partners recommendations. . In addition, kinetic degradation models will be developed for all microbes discussed above. In addition, microbial tailing and shoulder effects will be studied.A flow-through bench UV reactor will be developed to study and understand UV dose delivery. The team is also develop a sizing computer based program to compute doses for various microbes based on the D10 values Task 3.3: Developing chemical actinomters for UV dose verification AFC (industrial partner) is working with a range of liquid food companies to evaluate the applicability of UV treatment for disinfection. These firms require confirmation that the desired UV dose has been applied to their liquid food material. Our team would develop a chemical actinometer which could be used for UV dose verification. AFC will contribute to the development of the chemical actinometer. Task 3.4 Validation of UV dose by biodosimetry and chemical actinometry UV dose validation test will be conducted using bacteriophages and chemical based actinometers (generally recognized as safe by FDA) for a range of selected liquid foods. The tests will be conducted using a CB unit. Task 3.5 Develop a flow-through UV system for microbial inactivation studies Custom flow-through UV system will be built and validated. This unit will be designed to apply uniform, measurable UV irradiation field to a specific volume of fluids. A computer based program (sizing algorithm)will be developed to calculate, UV dosage and flow-rate based on the UV senstivity of the target microbe.

Impacts
What was accomplished under these goals? Objective 2 & 3. Collimated beam System Development and validation A novel optical method using a single- integrating sphere was developed to quantify the absorption, scattering, scattering anistrophy of highly scattering and absorbing liquid foods. Protocols for optical properties validation were also developed including UV fluence verification using a MS2 (a single-stranded RNA virus, a single-stranded RNA virus). A collimated beam system operating at 254 nm wave-length was designed and validated during this reporting period. This apparatus was designed to provide uniform, quantified irradiation to liquid samples, and the associated methods, including calibration, fluence determination, and quality assurance protocols, have been developed and standardized in the field of water disinfection. To enhance mixing, the irradiated volumes were reduced to 5 mL samples in 10 mL beakers, with continuous stirring. UV fluence values were calculated using the standard method based on measured UV irradiance and optical properties of the fluid. The collimated beam approach is based on using well-mixed samples in a quantifiable UV radiation field, accounting for all relevant optical factors such as beam divergence, reflection, and non-uniformity. It assumes that the mixing is sufficient to ensure that all fluid elements are exposed equally and that the geometric average UV intensity may therefore be used to calculate total fluence. This approach overcomes the problems in other studies in which stagnant, highly absorbent fluid samples were irradiated. Biodosimetry Studies In order to determine the actual UV fluence values delivered to an opaque fluid (i.e. apple juice), a viral clearance test was validated and conducted using a challenge organism using MS2, a single-stranded RNA virus, with an icosahedral shell approximately 27 nm in diameter. The UV sensitivity of the MS2 was first established using an inactivation test. MS2 bacteriophage was suspended in phosphate buffered saline, pH 7.1 and irradiated in a collimated beam to different UV fluence values. The buffer used in this characterization had a high transmittance at 254 nm, typically 90% at 1 cm path-length, so that the UV intensity gradient in the fluid was small, and therefore the uncertainty in the UV fluence was small. The 254 nm irradiance at the sample surface was measured using a radiometer with NIST-traceable calibration. The UV fluence gradient caused by optical absorbance of the fluid, along with the effects of beam divergence, non-uniformity, and surface reflection were all accounted for as recommended in the standard method. With the UV-sensitivity of the organism determined, the organism was then used to quantify the UV dose delivered to a UV-absorbing fluid. Ideally, the MS2 would be inoculated into the juice, followed by UV irradiation. However, the MS2 would be inactivated by the low pH of apple juice. For this reason, a surrogate fluid with the same optical absorbance and near-neutral pH was used to verify UV dose delivery. UV dose applied to the surrogate fluid was determined by using MS2 as the dose indicator. This is sometimes denoted as the reduction equivalent dose (RED) or fluence, since it is inferred from the log reduction of a well-characterized challenge organism. MS2 from the characterized population was inoculated into a surrogate fluid consisting of an aqueous solution of humic acid formulated to have the same optical absorbance as apple juice. The log reduction in numbers of active MS2 was used to calculate the UV dose received by the organic humic acid in each irradiation by using the sensitivity of the MS2 as established by the buffer tests. This relationship was used to determine the UV fluence values used in subsequent irradiations of apple juice. This test confirmed that doses from 0- 150 mJ·cm-2 can be applied to highly opaque liquid foods such as apple juice. A biodosimetry protocol was developed in this reporting period (workpackage 2 &3). Bacterial and Viral inactivation study Dedicated bacterial inactivation steps are essential to minimize the risk of foodborne infections in liquid food products. UV-C irradiation is an effective means of inactivating many pathogenic organisms and is used extensively in water treatment. It acts directly on nucleic acids of the target microbe and impedes its replication. UV irradiation might be an effective method of inactivating food borne pathogens in liquid foods. In this study, effective UV dose delivery was evaluated and validated in a range of fluids of varying optical properties (0.01 to 50 %/cm) at 253.7 nm wave-length. UV inactivation kinetics of two surrogate viruses in phosphate buffered saline and coconut water were studied using a collimated beam system. MS2 a single-stranded RNA virus, and T1UV a double-stranded DNA virus were selected as challenge micro-organisms. UV sensitivity (D10- mJcm-2) of MS2 and T1UV were quantified using a bioassay test. Target doses from 0 - 80 mJcm-2 were applied in this study. It very effectively inactivated the viral particles in the test fluids, confirming accurate dose delivery. The experiments also clearly demonstrated that 'dose is dose', regardless optics of fluid optics, the fluence-based inactivation rate constant would be identical. Against increasing demand for higher security against bacterial and viral contamination in liquid foods, UV irradiation inactivated viruses effectively (5 log removal). Optics of the fluids were the key controlling parameters and kinetics are invariant with fluid type. Data from this study enabled the understanding of UV treatment and accurate UV dose delivery in highly opaque fluid. Mycotoxins detoxification studies Patulin, a mycotoxin produced by several genera of fungi, is an important concern in apple-based products due to its toxicity and health consequences. Ultraviolet (UV) irradiation is a method of disinfection that is effective against many microorganisms and is also applied for chemical contaminant treatment via direct photolysis and advanced oxidation with sensitizers such as hydrogen peroxide. The present study evaluates whether UV could be an effective method to destroy patulin in apple juice. UV irradiation experiments were conducted using a collimated beam system operating at 253.7 nm. It was observed that patulin was not destroyed in pure water, but was degraded in pure and diluted apple juice upon UV irradiation, suggesting that chromophores (such as riboflavin) in the juice were important factors in the photo degradation. From an initial patulin concentration of approximately 200 ppb, UV dose of 400 mJ. cm-2 successfully reduced patulin concentration by 69.47 (±0.69) % (p<0.05). In cell culture studies, our results showed that increasing UV dose decreased the patulin-induced cytotoxicity in human peripheral blood cells. Cell viability percentage increased from 47.3% to 81.64% as UV dose increased from 0 to 400 mJ. cm-2. These doses would also achieve significant inactivation of viruses, bacteria, and many molds and fungi. This study clearly shows the potential for using UV treatment for patulin degradation in turbid liquid foods such as apple juice. Further studies on understanding the degradation mechanism of patulin by UV irradiation is required. The findings in this study serve as a demonstration of the changes that can occur patulin via UV treatment. While these results cannot be generalized to other juices, this study provides insight into the effects of UV-sterilization in liquid food treatment for patulin removal.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Islam, M.S., Patras, A., Pokharel, B., Wu, Y., Vergne, M.J., Shade, L., Xiao, H. & Sasges, M. (2016). UV-C irradiation as an alternative disinfection technique: Study of its effect on polyphenols and antioxidant activity of apple juice, Innovative Food Science and Emerging Technologies (2016), doi: 10.1016/j.ifset.2016.02.009
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Chandra, S., Patras, A., Pokharel, B., Bansode, R. R., Begum, A., Sasges, M. (2015). Patulin degradation and cytotoxicity evaluation of UV irradiated apple juice. Innovative Food Science and Emerging Technologies (Manuscript ID: IFSET-D-16-00214
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: The effect of UV irradiation on nutritional quality and cytotoxicity of apple juice. Journal of Agricultural and Food Chemistry
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Co-authored publication, titled Advancements in UV Treatment of Liquid foods: A Dose Delivery Study. 12th International Conference of Engineering and Food, Quebec, Canada, June 14-18th, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Co-authored publication, titled Application of Low Wave-length UV-C Irradiation for Treating Mycotoxins in Turbid Fluids. International Association of Food Protection, Oregon, Washington, July 25  28th, 2015
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Co-authored publication, titled UV-C Inactivation of Bacteria and Viruses in Coconut Water. International Association of Food Protection, Oregon, Washington July 31st  August 3rd, 2016 (accepted for presentation)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Co-authored publication, titled Effect of Ultraviolet-C irradiation and photosensitizers on mitigation of aflatoxins. IFT Annual Meeting + Food Expo, Chicago, July 16-19th , 2016 (accepted for presentation)