PROGRAM. Robert E. Brackett, Ph.D., IIT Vice President and IFSH Director

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1 SAT Ninth Annual Graduate Student Seminar Day Day One, Monday, April 16th, 2018 Moffett Campus, Building 91- Room 216 PROGRAM 9:30-9:40 9:40-10:00 Welcome and Opening Remarks Robert E. Brackett, Ph.D., IIT Vice President and IFSH Director Improved Sampling Methods for Detection of Food Allergens on Food-Contact Surfaces Magdalena Naziemiec, 10:00-10:20 Effectiveness of Push-through Cleaning Methods for Removing Milk Chocolate from a Stainless Steel Pipe and Butterfly Valve Liyun Zhang, 10:20-10:40 Seafood Allergen Cross-Contact Risk in Shared Fryers Anirudh Kaja, 10:40-10:50 BREAK 10:50-11:10 Effect of ph on Desiccation Survival on Salmonella Anatum Fangyu Chen, 11:10-11:30 11:30-11:40 Survival of Listeria monocytogenes on Sunflower Seeds During Storage Alisha Aggarwal, Closing Remarks Jason Wan, Ph.D., IFSH Associate Director 1

2 Magdalena Naziemiec Improved Sampling Methods for Detection of Food Allergens on Food-Contact Surfaces ABSTRACT: Total protein or immunochemical tests are essential to food manufacturers to determine if allergens remain on processing equipment after cleaning. Currently, there is no uniform environmental sampling protocols for allergens. This project was designed to optimize the amount of allergens recovered from stainless steel surfaces during swabbing as measured with the bicinchoninic (BCA) protein assay. This study evaluated the impact of swab material (rayon, polyester, foam), number of swabs (one, two, three), phosphate buffered saline (PBS) swabbing buffer with and without 0.05%- 1% detergent (Tween 20, 40, 60, 80) and commercial swabbing buffer on recovery of allergens applied to a stainless steel surface with and without heat. Solutions (0.5 ml; 1000 ppm or 2500 ppm) of whole egg powder, peanut butter and nonfat dry milk (NFDM) were pipetted directly onto stainless steel plates or directly onto swab heads. Plates were not dried, dried at ambient temperature, or with heat (65 C, 80 C; 30 or 60 minutes) and then sampled for allergens using swabs wetted in the different swabbing buffers. PBS with 0.05% Tween 20 (PBST) was used to extract protein from swab heads, which was then analyzed for protein content with the BCA assay. Conditions that gave optimized protein recoveries were then tested for allergen recovery with allergen-specific enzyme-linked immunosorbent assays (ELISAs). Experiments were conducted in triplicate. In general, higher total protein recoveries were measured for allergens pipetted directly onto swabs composed of rayon than other materials. Rayon also recovered more protein when allergens were pipetted onto plates. After swabbing unheated surfaces, recoveries for peanut [rayon, 1000 ppm: 25% (11% CV)] were similar or lower than for milk [26% (3% CV)] or egg [37% (12% CV)]; similar recoveries were obtained regardless of heating or allergen stock concentration. Overall, recoveries did not decrease with increased heating conditions. More protein was recovered with three rayon swabs [2500 ppm liquid NFDM: 48% (1% CV)] than with one rayon [32% (3% CV)] or two rayon [46% (4% CV)] swabs, however there was no large difference between the recovery of two and three swabs. For one swab, the highest protein recoveries were measured with PBST swabbing buffers and the lowest were measured with PBS. However, when plates were sampled with two rayon swabs, there was little difference in BCA or ELISA recovery between PBST, PBS, or commercial swabbing buffer. Environmental sampling evaluates the effectiveness of cleaning protocol and informs decisions made by food manufacturers. This project will provide information on best environmental sampling practices to improve allergen detection. 2

3 Liyun Zhang Effectiveness of Push-through Cleaning Methods for Removing Milk Chocolate from a Stainless Steel Pipe and Butterfly Valve ABSTRACT: Dark chocolate is considered a high-risk food for milk-allergic consumers when manufactured on processing lines also used to produce milk chocolate. Inadequate cleaning of shared chocolate processing lines can result in milk contamination of dark chocolate products. A study evaluated the effectiveness of push-through cleaning methods for removing milk chocolate from a contaminated stainless steel pipe and butterfly valve. Melted milk chocolate ( g, 85,000 ppm milk) was used to coat inner surfaces of a heated (40ºC) standard (1.5 OD) sanitary stainless steel pipe (30.5 cm length) and an attached butterfly valve. Two scenarios were studied: 1) milk chocolate-contaminated pipe and valve and 2) milk chocolate-contaminated pipe and clean valve. Milk-free dark chocolate (~27 kg, 40 C) was pumped (~140 g/sec) through the various combinations of milk chocolate-contaminated pipe and valve to evaluate two cleaning methods: 1) no cleaning and 2) use of a silicone pig (7.6 cm length) to purge milk chocolate from the pipe. Dark chocolate samples (~300 g) collected at 7-sec intervals were homogenized and analyzed for milk concentrations by ELISA (Neogen Veratox for Total Milk). Experimental trials and ELISA analyses were conducted in triplicate. Dark chocolate push-through alone for the contaminated pipe and valve resulted in initial milk levels of ~3200 (3.4% CV)-6000 (10% CV) ppm milk in samples obtained within the first few seconds of collection. After kg of dark chocolate was pumped through the contaminated pipe and valve, milk levels were below the ELISA limit of quantitation (LOQ; 2.5 ppm). Use of the pig dramatically reduced the initial levels of milk in dark chocolate samples for the experiment with the contaminated pipe and valve [110 (7.6% CV)-180 (16% CV) ppm milk]. For this experiment, 18 kg of dark chocolate push-through the system was required to obtain milk levels < LOQ. In contrast, use of the pig in the contaminated pipe/clean valve resulted in initial milk levels <LOQ. Utilizing a pig in a cleaning regimen was found to be effective in removing milk chocolate from the contaminated pipe. However, the butterfly valve was difficult to clean and the primary contributor for the introduction of milk in dark chocolate as a result of cross-contact. This study aids in development and assessment of pushthrough and other cleaning procedures for preventing allergen cross-contact. 3

4 Anirudh Kaja Seafood Allergen Cross-Contact Risk in Shared Fryers ABSTRACT: Consumption of minute amounts of seafood allergens can cause anaphylactic reactions in individuals with seafood allergies. Many restaurants use the same fryer to cook various types of foods including seafood and vegetable products. This study investigated the transfer of crustacean allergens into oil and cross-contact to a secondary food (French fries) cooked in the same oil. Ten, 100-g batches of black tiger shrimp were fried in soybean oil (4.7 L) for 3 minutes at 176 C, and samples of frying oil collected immediately after each batch of shrimp was fried. Subsequently, four, 100-g batches of French fries were fried in the shrimp-contaminated oil at 176 C for 3 minutes, and samples of fries collected after frying. Oil and French fry samples were analyzed using two shrimp-specific ELISA kits. Tropomyosin, the major shrimp allergen, was measured using the ELISA Systems assay and crustacean protein was measured with the more sensitive Maruha Nichiro ELISA test. The Maruha Nichiro ELISA kit measured ppm (1.9% CV) ppm (2.7% CV) crustacean protein in the oil samples after frying up to ten batches of shrimp. The ELISA Systems kit detected 0.09 ppm (12% CV) ppm (4.2% CV) tropomyosin. Total protein content of the shrimp-contaminated oil samples was measured using the BCA-RAC assay, which showed protein concentrations of 9.82 ppm (3.2% CV) ppm (10.9% CV). Levels of tropomyosin in French fries and oil samples obtained after frying the French fries were below the limit of quantitation (LOQ; 0.05 ppm) of the ELISA System kit. However, the more sensitive Maruha Nichiro kit detected crustacean protein in oil samples after frying French fries, at concentrations ranging from 0.10 (4.1% CV) 0.84 ppm (2.6% CV). French fries contained 0.82 ppm (6.0% CV) ppm (5.1% CV) crustacean protein, due to cross-contact that occurred during frying. The results of this study suggest that low levels of crustacean proteins are transferred to oil and to a secondary food matrix in shared fryers. 4

5 Fangyu Chen Effect of ph on Desiccation Survival on Salmonella Anatum ABSTRACT: One intrinsic characteristic of low-moisture foods frequently overlooked is ph. Although ph is known to affect the survival of microorganisms in high moisture foods, its influence in low-moisture foods has not been examined. This study investigates the effect of growth at different ph on the survival of Salmonella during desiccation under varied ph conditions. Salmonella Anatum 6802 was grown in Tryptic Soy Broth at ph 7 (neutral) for 24±2 h at 37 C. 24 h culture (0.1mL) was spread on neutral ph trypticase soy agar with 0.6% yeast extract (TSAYE) plates and harvested with approximately 1 ml of neutral ph buffered peptone water (BPW). The harvested suspension (1 ml) was added to 9 ml buffer adjusted to ph 3, 4, 5, 7, and 8 and held for 30 min, spotted onto a cellulose filter and dried in a biohazard cabinet (23±2 C) overnight (12±2 h). In addition, harvested cells were first dried on cellulose filters then soaked for 30 min in 10 ml buffer adjusted to ph 3, 4, 5, 7, and 8. All were enumerated on TSAYE with 0.005% ferric ammonium citrate and 0.03% sodium thiosulfate (Modified TSAYE). Additionally, cells were grown on TSAYE adjusted to ph 4, 5, 7, or 8. After 24±2 h incubation, the lawn was harvested with buffer at the same ph as on which it was grown and desiccated as before. Salmonella was enumerated as before. Desiccated cells grown at neutral ph subsequently exposed to ph 3, 4, 5, 7, and 8 buffer prior to desiccation resulted in reductions of 2.55±0.14, 0.32±0.65, 0.18±0.26, 0.30±0.03 and 0.01±0.30 log CFU/mL, respectively. Differences between exposure at ph 3 and higher ph were significant (p<0.05). Non-adapted cells displayed a similar trend with average counts approximately 1 log CFU/mL lower. Cells grown on TSAYE with ph adjusted to 4, 5, 7, or 8 did not yield significantly different rates of survival when subsequently desiccated. Acid adapted cells survived at significantly higher levels than non-acid adapted cells when desiccated at low ph. When conducting challenge studies with acidic low-moisture foods acid adapted cells should be used. 5

6 Alisha Aggarwal Master of Science Degree Candidate, Food safety and Technology Survival of Listeria monocytogenes on Sunflower Seeds during Storage ABSTRACT: Recently sunflower seeds have been recalled due to the presence of Listeria monocytogenes. At present, little is known about the survival of Listeria monocytogenes during processing and storage of sunflower seeds. The objective of this study was to determine the survival rate of Listeria monocytogenes on raw in shell kernels, raw shelled kernels and oil-roasted sunflower kernels during storage at varied temperature and humidity. Sunflower seeds (raw inshell kernels, raw shelled kernels and oil-roasted kernels) were inoculated at a targeted 6-log CFU/g level with a cocktail of 6 strains (Scott A, ATCC 13932, ATCC 49594, NZRM 4242, NZRM 4237, FRRB 02542) of Listeria monocytogenes, followed by air drying at ambient temperature for 24 hours. Seeds were then transferred into storage at 10, 25 and 37 C at 40% and 70% relative humidity (RH). Samples were periodically removed from storage for determination of water activity (aw) and microbial population. To enumerate populations, samples were serially diluted in buffered peptone water and plated on tryptic soy agar with yeast extract. Listeria monocytogenes was confirmed by plating on modified oxford agar. Microbial population on raw in-shell kernels decreased significantly from 6.9 ± 0.57 log CFU/g to 0.93 ± log CFU/g (p=0.0003) and from 6.4 ± 0.36 log CFU/g to 0.43 ± 0.75 log CFU/g (p=0.0001) at 37 C 70% and 40% RH respectively in 14 days. To achieve a similar reduction at 25 C required 28 days. Differences in decline based on temperature and RH were significant (p= ). No significant decreases were observed when stored at 10 C for 28 days (p>0.05). The results indicate that Listeria monocytogenes is capable of surviving for extended periods at low temperature. However, survival during storage is affected by both temperature and RH. 6