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INVESTIGATOR: Patras, A.

PERFORMING INSTITUTION:
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE, TENNESSEE 37209

AN INTEGRATED APPROACH TO CONTROL FOODBORNE BACTERIAL, VIRAL, AND MYCOTOXINS IN HIGH RISK FOODS USING ULTRAVIOLET TECHNOLOGIES

NON-TECHNICAL SUMMARY: This project, integrating research with education activities, addresses the USDA priority area "Food Safety" by developing a novel continuous flow-through Ultraviolet Light (UV) based system for non-thermal pasteurization of liquid foods. Research in UV technology for disinfection of liquid foods in general is very limited and not fully understood, and particularly scarce for disinfection of highly absorbing and scattering liquid foods (milk, flavored water, juices with particulates and high concentration of proteins and other polymers). To improve this situation, engineering solutions are needed to remove several limitations of the technology, scientific research is necessary to understand the effects of the UV process on nutritional value, safety quality, and the cost and benefit concerns with implementation of the technology in new plants and retrofitting in existing plants must be addressed. Industrial implementation of these novel technologies will help food companies to be in compliance with the FDA Food Safety Modernization Act (FSMA). This project will also reduce technical and regulatory hurdles for food industries, particularly small and medium sized companies, to adopt the novel technologies for the production of microbiologically-safe and high-quality beverages. Inappropriate and insufficient decontamination have led to numerous viral and bacterial outbreaks in the past. This project will address these challenges by developing a novel flow-through UV reactor and expansion of this technology into new regimes. The project also intends to develop formal education programs at graduate and under-graduate levels as well as nonformal education programs for broader audiences on new food safety technologies.

OBJECTIVES: A.This program is designed in a series of logical, sequential work packages, supported by NCAT and industrial collaborators, Aquafine Corporation (AFC) and Trojan Technologies (TUV) and Vita Coco, VC. The members of this program with expertise in complementary academic multi-disciplines will work closely to achieve 7 key objectives. These 7 objectives are structured into 7 work packages (WP). It is important to note that the WPs are all highly interconnected. In addition most WPs involve the cooperation of various scientists, SMEs, and an education specialist for prototyping and validation.Specific objectives (1-7)1. Perform effective management of all project work and resources; 2. Conduct extensive engineering studies to optimize design of a novel pilot UV-C reactor, and assist in scaling-up of UV assisted pasteurization systems; 3. Evaluate, improve maximize, and disseminate findings concerning safety of UV processed foods; 4. Perform chemical profiling and cytotoxicity analysis of UV irradiated fluids in cell culture and animal models; 5. Conduct accelerated storage and sensory studies for UV processed liquid foods; 6. Develop, validate, and disseminate UV processes for selected liquid food products for FDA FSMA compliance; 7. Develop formal education programs at graduate and under-graduate levels as well as nonformal education programs for broader audiences;

APPROACH: Consumer education: An educational package for consumers will be developed and implemented the third year of the project. Materials developed for consumers will be based on the findings from this project. Consumers will be informed in simple terms about the UV-C technology, its benefits and safety. A simple brochure will be placed on the TSU College web-site, and collaborator Godwin's food safety website. It will be promoted through the University's Public Relations Office, as well as on the College's Facebook page. Each week for three months a tweet will be sent out on twitter. Reminder messages will also be posted on Facebook, each featuring a different facet of the UV-C technology.

PROGRESS: 2019/03 TO 2020/03
Target Audience:Target audiences reached by this project includes reactor manufacturers and designers, regulatory agencies, food companies (Unique Food solutions, Trojan technologies, Aquafine Corporation, Vita Coco etc), faculty (research, extension) and graduate students. Changes/Problems:The team lost two graduate students, they passed away in an accident. The loss was enormous. What opportunities for training and professional development has the project provided?This research project has supported the research training of 2 graduate students (1 postdoctoral scholar). Training is still undergoing. 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?The results have been communicated and were presented at IFT and IAFP conferences. Knoelwledge transfer sessions were conducted with industrial stake-holder group. What do you plan to do during the next reporting period to accomplish the goals?The team would expand on cytotoxicity of UV irradiated foods. The group will perform chemical profiling and cytotoxicity analysis of UV irradiated fluids in cell culture and animal models. In addition, the group will conduct accelerated storage and sensory studies for UV processed liquid foods.

IMPACT: 2019/03 TO 2020/03
What was accomplished under these goals? Ultraviolet (UV) light has been used for decades for disinfecting water, and is broadly applied in Europe and North America. But until recently it has not been adopted for opaque fluids such as liquid foods and beverages including milk. Recently, successful application in juice and milk treatment has demonstrated the feasibility of UV for treating these fluids, and UV technology has started to emerge as a promising non-thermal preservation process. As a non-thermal, non-chemical disinfection technology, UV is anticipated to have minimal effects on product quality, flavor, and nutritive content. UV treatment is effective against food and water borne pathogens, spoilage microflora, spores, and can control pathogen levels to comply with regulatory requirements. The challenge remains that the range of optical and other properties of beverages is extremely broad. Also, each disinfection process may have different microbiological targets, meaning that each UV process has to be developed individually using specific system designs. In each application, three factors must be assessed: the treatment level required for the necessary reduction in target pathogen levels; the impact on product quality; and the regulatory requirements. Three different cases studies were conducted. Case study 1: Treatment of whole milk (WM) by UV-C irradiation: a biodosimetry and volatile profile analysis. The objective of this study was to determine the feasibility of UV to maximize the level of protection from contamination while maintaining sensory quality. All doses/ fluence were verified using T1 bacteriophage (data not shown). B. cereus ATCC 14579 and E. coli ATCC 25922 was chosen as microbial targets. Known concentration of spore or vegetative cells were spiked in WM and irradiated at different fluence levels. Separate set of experiments were conducted for volatile profiling. Prior to any inactivation studies, preliminary studies will be carried out to estimate the D10 values of the target microbes. All tests will be carried in buffer (phosphate buffer saline) using a batch collimated beam (CB) system operating at 254 nm wave-length. Microbial inactivation studies were carried out using a flow-through UV system operating at 254 nm wave-length. Various sets of fluence was delivered to WM depending on the microbe and its D10 value. 7.1 log reduction of E. coli was observed at UV fluence of 27 mJ cm-2 (p<0.05). D10 value was observed to be 3.77 mJ. cm-2. It is quite apparent from the data that E. coli inactivation followed first order R2 = 0.99. 4.2 log reduction of B. cereus was observed at UV fluence of 41.8 mJ cm-2 (p<0.05). D10 value was observed to be 9.46 mJ. cm-2. The volatile aroma compounds profile in WM was analyzed using electronic nose. The results revealed that esters, aldehydes, alcohols and ketones were accounted for proportion of the volatile compounds. 1-Propanol, 2-methyl-, 2-methylpropanal and ethyl 2-methylbutyrate were identified as major volatile compounds in untreated WM. In comparison to untreated samples, as UV fluence increased, the proportion of aldehydes, alcohols and ethers decreased in total volatiles. In contrast, an absolute increase in other compounds esters, ketones and acids (from 32 mJ. cm-2) was observed. It was observed that comparative analysis between UV-C treated samples revealed that the proportion of esters, aldehydes, alcohols and ketones in total volatiles were slightly altered. In addition, low energy requirement makes UV-C treatment as a potential alternative process to existing thermal pasteurization for whole milk treatment. The novelty of this study is the development of continuous UV system based on dean flow pattern to treat WM with high absorbance. Case study 2: This study investigates the effectiveness of UV-C irradiation on the PPO and POD activity of coconut water. In addition, we report the effect of UV-C irradiation on the concentration of free essential amino acids (histidine (His), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), leucine (Leu), and isoleucine (Ile)) and sensory quality of coconut water. The impact of ultraviolet light (UV-C) irradiation on oxidative enzymes [Polyphenol oxidase (PPO) and Peroxidase (POD)], free essential amino acids and sensory profile of coconut water were investigated. PPO and POD activities were lost to 94 and 93%, respectively of its original value at fluence level of 400 mJ/cm2. Inactivation kinetics of both enzymes were fitted to nonlinear Weibull model with an increase in UV dosage with a high coefficient of determination (R2 > 0.97) and low root mean square error (RMSE < 0.06). No significant change was observed in all essential amino acids (p [ 0.05) after UV-C treatment up to maximum delivered fluence of 400 mJ/ cm2. Sensory attributes of coconut water up to a treated UV-C fluence level of 200 mJ/cm2 were well retained in terms of chosen descriptors (p [ 0.05). This study allow to further investigate the development of UV-C light technology for inhibition of spoilage enzymes and prolonged shelf-life of low acid beverages. Case study 3. This study was carried out to determine the degradation kinetics and the possible degradation mechanism of aflatoxins (AFB1& AFM1) in a transparent matrix like ultrapure water and WM, without the hindrance of other biomolecules which may result in UV attenuation. This study also assesses the cytotoxicity of UV-A treated samples which contain residual AFB1 and AFM1 and their degradation products in water using human hepatoma cell line (HepG2). Irradiation experiments were conducted using an LED system operating at 365 nm (monochromatic wave-length). Known concentrations of aflatoxins were spiked in water and irradiated at UV doses ranging from (0 - 1200 mJ/cm2). The concentration of AFB1 and AFM1 was determined by HPLC with fluorescence detection. LC-MS/MS product ion scans were used to identify and semi-quantify degraded products of AFB1 and AFM1. It was observed that UV irradiation significantly reduced aflatoxins in pure water and WM. In comparison to control, at dose of 1200 mJ/cm2, UV-A irradiation reduced AFB1 and AFM1 concentrations to 70 ± 0.27 and 84 ± 1.95 %, respectively. We hypothesize that the formation of reactive species initiated by UV-A light may have caused photolysis of AFB1 and AFM1 molecules. In cell culture studies, our results demonstrated that the increase of UV-A dosage decreased the aflatoxins-induced cytotoxicity in HepG2 cells, and no significant aflatoxin-induced cytotoxicity was observed at UV-A dose of 1200 mJ/cm2. Results from this study will be used to develop UV-A dose response curves in milk, vegetable oils, and other liquid foods. To summarize, the application UV technology for food allows moving from traditional pasteurization to non-thermal UV disinfection that has significant advantages for cost and energy savings while ensuring the safety and quality of foods. The team has developed a pilot UV system based on dean flow concept an currently validating on commercial flow-rates.

PUBLICATIONS (not previously reported): 2019/03 TO 2020/03
1. Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Stanley, J., Patras A., B Penyala, Bansode, R. R., Vergne, M.J. (2020)Performance of a UV-A LED system for degradation of Aflatoxins B1 and M1 in pure water: kinetics and cytotoxicity study
2. Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Yannam, S., Patras A.,. B Penyala, Gopisetty, V., R, Ramasamy (2020). Effect of UV-C irradiation on the inactivation kinetics of oxidative enzymes, essential amino acids and sensory properties of coconut water
3. Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Pendyala, B., Patras, A., Gopisetty, V., Sasges M., Michael Sasges, Balamurugan, S (2019). Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light. Foodborne Pathogens and Disease, https://doi.org/10.1089/fpd.2019.2623
4. Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Pendyala, B., Patras, A., Gopisetty, V., Sasges M., Michael Sasges. (2020). Evaluation of UV-C irradiation treatments on microbial safety, ascorbic acid and volatile aromatics content of watermelon beverage.Food and Bioprocess Technology, Food and Bioprocess Technology volume 13,101â111