Progress 09/01/18 to 08/31/22

Outputs
Target Audience:Oil Scientists:Dr. Martinez attended the 17th EuroFedLipid Conference and Expo in October 2109 in Sevilla, Spain and present a poster entitled, "FryCheckTM - A New, Robust Quick Test for Frying Oil Quality". Denis Callewaert as well as our collaborators John Everard and Susan Knowlton at CortevaTM Agriscience are co-authors on this poster which described the current status of studies of the FryCheckTM dipstick. These include its performance in comparison to FTNIR, Oleotest, 3M Free Fatty Acid test strips and Testo methods, in measuring the TPC content and overall quality of high oleic soy and canola frying oils, as well as assay performance as a function of temperature. Since the TPC levels in frying oils are regulated in Europe, this conference was highly productive and provided the opportunity to connect and work with regulators and potential customers that need a reliable quick test in order to remain in compliance with EU frying oil regulations. Dr. Martinez met with several oil scientists, manufacturers, and distributions, and gained a better understanding of the European edible oil market? Oil Producers & Distributors:In 2020, Denis Callewaert, the CEO of Oxford Biomedical presented an hour-long webinar on the measurement of fry life and the benefits of using FryCheck to a large group of Asian edible oil producers sponsored by the US Soy Export Council (USSEC). The USSEC and its associates have continued to request samples for evaluation, pending EPA approval. Restaurant owners & Operators: Dr. Martinez and two other OBR employees attended the 2018 National Restaurant Association Show in Chicago at which our company had a booth (supported by a grant from the State of Michigan). There was great interest in FryCheck - over 500 attendees signed up to receive more information and to evaluate FryCheck when it was ready for commercial launch. The 2020 and 2021 NRA shows were cancelled due to the COVID pandemic. Anticipating EPA approval, we exhibited at the 2022 NRA show. This would have been the official "rollout" of a FryCheck prototype to customers who were willing to take part in our first beta evaluation. Over 200 participants requested further information but, without EPA approval, we are still unable to provide samples to most interested restaurants.? Changes/Problems:Changes/Problems Test Strip Characteristics: Many formulations were evaluated in order to provide the best response to oil polarity and to overcome early problems with a short shelf life. Use of higher grade chemicals and a second recryatallization of the dye improved reproducibility and performance. A major source of concern was ultimately found to be the acrylic acid in the adhesives commonly used for dry chemistry dipsticks. Multiple non-acidic adhesives were evaluated, but none of them provided the needed mechanical stability needed. Ultimately, we elected to use two pieces of one-sided tape to hold the test pad onto the plastic backing. Effect of Oil Type: Although FryCheck performed as intended for multiple soy, Canola, Palm and Peanut oils, FryCheck responded slower to hydrogenated soy oil, it responded more rapidly to Plenish than commodity soy. These results suggest that we may ultimately need to modify our formulation for optimal results with such oil types. A pilot study performed at a very large agribusiness company showed that frying raw, uncoated/unseasoned chicken wings created a situation where rendering of the chicken fat was occurring. This caused the overall volume of the oil to increase during frying, which is usually opposite of what happens as cooks need to add or "top off" the oil during frying. FryCheck strips that were used to test this oil as the turned dark blue/grey, even when the oil was obviously unfit to consume, giving a false "good oil" response. We determined that the cause of this pad color anomaly was from the chicken fat which was effectively diluting the soybean oil. We are still waiting for the EPA to complete review of our PMN and still can't release test strips to potential customers - with the exception of those who have a research grade test kitchen.? What opportunities for training and professional development has the project provided?In 2019, Dr. Martinez travelled to the PortaScience facility in Moorestown, NJ review and trouble-shoot the strip manufacturing. During this visit, the coating, drying and mounting procedures were performed, with some improvements being made during the two pilot runs that were performed during the visit? How have the results been disseminated to communities of interest?Dr. Martinez attended the 17thEuroFedLipid Conference and Expo in October 2109 in Sevilla, Spain and present a poster entitled, "FryCheckTM - A New, Robust Quick Test for Frying Oil Quality". Denis Callewaert as well as our collaborators John Everard and Susan Knowlton at CortevaTM Agriscience are co-authors on this poster which described the current status of studies of the FryCheckTM dipstick. These include its performance in comparison to FTNIR, Oleotest, 3M Free Fatty Acid test strips and Testo methods, in measuring the TPC content and overall quality of high oleic soy and canola frying oils, as well as assay performance as a function of temperature. Since the TPC levels in frying oils are regulated in Europe, this conference was highly productive and provided the opportunity to connect and work with regulators and potential customers that need a reliable quick test in order to remain in compliance with EU frying oil regulations. Dr. Martinez met with several oil scientists, manufacturers, and distributions, and gained a better understanding of the European edible oil market? What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Complete Development of FryCheckTMTest Strips for Commodity Soy and Canola Oils: The dye used in the FryCheckTM formulation is sensitive to the chemical modifications that occur during frying, so its response to frying oils with very different composition was expected to be slightly different. Given that commodity soybean and Canola oils, and frying oil blends with a high concentration of these oils are the predominant oils currently used for deep-frying in North America, our primary goal was to complete development of a commercial FryCheckTM product for this market, then to examine its performance with other oil types. Many formulations were evaluated in order to provide the best response to oil polarity and to overcome early problems with a short shelf life. A major source of concern was ultimately found to be the acrylic acid in the adhesives commonly used for dry chemistry dipsticks. Ultimately, we elected to use two pieces of one-sided tape to hold the test pad onto the plastic backing. The test strip was widened to 1.0 cm for better appearance, handling characteristics, and adhesion of the pad to the strip. Test strips produced using better grade reagents were stable for several months at elevates (37°C) temperatures without a desiccant. Objective 2:Test Strip Validation: The %TPC results obtained using FryCheckTM were compared to results obtained using the Official Method Cd 20-91, "Determination of Polar Compounds in Frying Fats" method (AOCS Cd 20-91), a tedious laboratory method that is the reference standard for %TPC. We performed head-to-head comparison with other commercial "quick tests" for monitoring frying oil, including: 3M® Free Fatty Acid strips; Testo® Dielectric Constant Meter; OleoTest® and the Miroil Test. For most samples, the results show a good correlation of FryCheck visual readings with AOCS values, although FryCheck is presently less robust than desired. Objective 3: Develop FryCheckTM products that are optimized for additional fry oils: In order to provide for calibration and validation, a series of oil samples with known total polar content were prepared by collecting samples at various times during frying - worked very well. For product development and standardization calibrators are needed that can be frozen and used to perform quality control on test strip lots during manufacturing. Whereas FryCheck responded slower to hydrogenated soy oil, it responded more rapidly to Plenish than commodity soy. These results suggest that we may ultimately need to modify our formulation for optimal results with such oil types. Objective 4:Determine the effect of food type on the performance of FryCheckTM: The types of foods and the frying process in restaurants differ in many aspects from very controlled laboratory fry studies. Therefore, in addition to expanding the types of foods tested in our own lab, we collaborated with several outside organizations to evaluate how FryCheck performed for them. Oil samples were collected from the frying vats during the experiment and analyzed with FTNIR for total polar compounds content, then measured with FryCheck test strips. This work demonstrated that the deterioration of the oil could be followed using FryCheck test strips throughout the frying process. However, A pilot study performed at a very large agribusiness company showed that frying raw, uncoated/unseasoned chicken wings created a situation where rendering of the chicken fat was occurring. This caused the overall volume of the oil to increase during frying, which is usually opposite of what happens as cooks need to add or "top off" the oil during frying. FryCheck strips that were used to test this oil as the turned dark blue/grey, even when the oil was obviously unfit to consume, giving a false "good oil" response. We determined that the cause of this pad color anomaly was from the chicken fat which was effectively diluting the soybean oil. Objective 5:Determine the utility of FryCheckTMto detect adulterated "new" frying oil samples. In developing parts of the world where the demand for cooking oil is increasing, waste cooking oil is sometimes added to new oil and resold, creating a mixture termed "gutter oil" to increase profits. It is estimated that up to 10% of the total volume of cooking oil consumed in certain areas of China is gutter oil. The FryCheckTM dipstick is sensitive to the different composition of new oils, and in preliminary experiments we have observed that adulterating fresh vegetable oil with a small percentage of heavily used oil is detectable by using a FryCheckTM prototype. Objective 6:Develop and test a mobile phone application for more accurate pad color reading: Visual comparisons the %TPC to the color scale on the product vial are somewhat subjective and vary between individuals. Thus , to achieve more accurate and precise readings of the %TPC values, we partnered with Spark Diagnostics, LLC. to adapt their proprietary, dipstick color reading technology to FryCheckTM strips. This technology involves using the cell phone camera and calibrator colors incorporated into the dipstick for orientation and normalization of colors relative to the lighting conditions and analysis of the test results. The app was found to reproducibly measure color changes in the TPC dry chemistry dipstick to measure TPC in frying oil with the corresponding color change from dark blue (fresh) to bright yellow/orange (>26%). Additional 6 gray calibrator lines were printed at varying gray values for the lighting conditions. Pattern recognition algorithms automatically correct for the tilt and other artifacts in the images such as blurriness due to user error. The standardized color response showed a linear correlation to the TPC concentration. Regression analysis to predict the TPC values for both iOS and Android systems showed a high predictive capability. The correlation coefficient >0.94 for all the models indicates a reliable prediction of TPC values. Further, the standard error of the prediction is in the range of 0.92 to 2.6% suggesting that the predictive values are much better than the color chart developed for visual readings of the dipstick (typically ~ +/- 6% units). Objective 7:Finalize high volume manufacturing, QA and packaging. The delamination of the pad from the surface of the plastic test strip has plagued this project from the start. We finally were able to eliminate tape containing acrylic acid and attach the test pad to the plastic backing by taping the top and bottom of the pad to the backing without adhesive on the back of the pad - resulted in a much more physically robust test that would not delaminate during shipping and handling. A short video tutorial is in preparation for uploading to the www.frycheck.com. EPA Regulatory Compliance, Pre-Manufacture Notice:Not included in our original Work Plan is the submission of a Pre- Manufacture Notice (PMN) to the Environmental Protection Agency for our proprietary dye. The blue dye used as the indicator in FryCheck cannot be manufactured and sold to the public in the United States without being listed on the Toxic Substances Control Act (TSCA) Chemical Substance Inventory. In June 2021 we applied for a Pre-Manufacture Notice (PMN) to allow for the manufacturing of our dye in up to kilogram quantities. This application has passed review by multiple EPA sections. However, as of this date, we are still awaiting final acceptance. Without this acceptance, we cannot make commercial quantities of the dye, nor are we permitted to have potential customers review our protype - except those few large organizations that have their own laboratory test kitchen.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: "FryCheckTM - A New, Robust Quick Test for Frying Oil Quality". Enrique Martinez1, Denis Callewaert1, Bev Krejsa2, John Everard2 and Susan Knowlton3. 1Food Quality Testing Corp, Rochester Hills, Michigan, United States. 2CortevaTM Agriscience, Johnston, Iowa, United States. 3CortevaTM Agriscience, Wilmington, Delaware, USA. Presented at at the 17th International Congress of the European Federation for the Science and Technology of Lipids (EuroFedLipid) on 10/20 to 10/23, 2019, in Sevilla, Spain.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience: Oil Scientists:Dr. Martinez attended the 17thEuroFedLipid Conference and Expo in October 2109 in Sevilla, Spain and present a poster entitled, "FryCheck™ - A New, Robust Quick Test for Frying Oil Quality". Denis Callewaert as well as our collaboratorsJohn Everard and Susan KnowltonatCorteva™ Agriscienceare co-authors on this poster which described the current status of studies of the FryCheck™ dipstick. These include its performance in comparison to FTNIR, Oleotest, 3M Free Fatty Acid test strips and Testo methods, in measuring the TPC content and overall quality of high oleic soy and canola frying oils, as well as assay performance as a function of temperature. Since the TPC levels in frying oils are regulated in Europe, this conference was highly productive and provided the opportunity to connect and work with regulators and potential customers that need a reliable quick test in order to remain in compliance with EU frying oil regulations. Dr. Martinez met with several oil scientists, manufacturers, and distributions, and gained a better understanding of the European edible oil market? Oil Producers & Distributors:In 2020, Denis Callewaert, the CEO of Oxford Biomedical presented an hour-long webinar on the measurement of fry life and the benefits of using FryCheck to a large group of Asian edible oil producers sponsored by the US Soy Export Council (USSEC).The USSEC and its associates have continued to request samples for evaluation, pending EPA approval. Restaurant owners & Operators:Dr. Martinez and two other OBR employees attended the 2018 National Restaurant Association Show in Chicago at which our company had a booth (supported by a grant from the State of Michigan). There was great interest in FryCheck - over 500 attendees signed up to receive more information and to evaluate FryCheck when it was ready for commercial launch.The 2020 and 2021 NRA shows were cancelled due to the COVID pandemic. Anticipating EPA approval, we exhibited at the 2022 NRA show.This would have been the official "rollout" of a FryCheck prototype to customers who were willing to take part in our first beta evaluation. Over 200 participants requested further information but, without EPA approval, we are still unable to provide samples to most interested restaurants. Changes/Problems:Test Strip Characteristics: Many formulations were evaluated in order to provide the best response to oil polarity and to overcome early problems with a short shelf life. Use of higher grade chemicals and a second recryatallization of the dye improved reproducibility and performance. A major source of concern was ultimately found to be the acrylic acid in the adhesives commonly used for dry chemistry dipsticks.Multiple non-acidic adhesives were evaluated, but none of them provided the needed mechanical stability needed.Ultimately, we elected to use two pieces of one-sided tape to hold the test pad onto the plastic backing. Effect of Oil Type: Although FryCheck performed as intended for multiple soy, Canola, Palm and Peanut oils,FryCheck responded slower to hydrogenated soy oil, it responded more rapidly to Plenish than commodity soy. These results suggest that we may ultimately need to modify our formulation for optimal results with such oil types.A pilot study performed at a very large agribusiness company showed that frying raw, uncoated/unseasoned chicken wings created a situation where rendering of the chicken fat was occurring. This caused the overall volume of the oil to increase during frying, which is usually opposite of what happens as cooks need to add or "top off" the oil during frying. FryCheck strips that were used to test this oil as the turned dark blue/grey, even when the oil was obviously unfit to consume, giving a false "good oil" response. We determined that the cause of this pad color anomaly was from the chicken fat which was effectively diluting the soybean oil. We are still waiting for the EPA to complete review of our PMN and still can't release test strips to potential customers - with the exception of those who have a research grade test kitchen. What opportunities for training and professional development has the project provided?In 2019, Dr. Martinez travelled to the PortaScience facility in Moorestown, NJ review and trouble-shoot the strip manufacturing. During this visit, the coating, drying and mounting procedures were performed, with some improvements being made during the two pilot runs that were performed during the visit How have the results been disseminated to communities of interest? Dr. Martinez attended the 17thEuroFedLipid Conference and Expo in October 2109 in Sevilla, Spain and present a poster entitled, "FryCheck™ - A New, Robust Quick Test for Frying Oil Quality". Denis Callewaert as well as our collaboratorsJohn Everard and Susan KnowltonatCorteva™ Agriscienceare co-authors on this poster which described the current status of studies of the FryCheck™ dipstick. These include its performance in comparison to FTNIR, Oleotest, 3M Free Fatty Acid test strips and Testo methods, in measuring the TPC content and overall quality of high oleic soy and canola frying oils, as well as assay performance as a function of temperature. Since the TPC levels in frying oils are regulated in Europe, this conference was highly productive and provided the opportunity to connect and work with regulators and potential customers that need a reliable quick test in order to remain in compliance with EU frying oil regulations. Dr. Martinez met with several oil scientists, manufacturers, and distributions, and gained a better understanding of the European edible oil market What do you plan to do during the next reporting period to accomplish the goals?Once we have EPA approval, we are ready to rapidly provide samples, improve the product as/if needed, and to market it globally through multiple channels.

Impacts
What was accomplished under these goals? Objective 1: Complete Development of FryCheck™Test Strips for Commodity Soy and Canola Oils:The dye used in the FryCheck™ formulation is sensitive to the chemical modifications that occur during frying, so its response to frying oils with very different composition was expected to be slightly different. Given that commodity soybean and Canola oils, and frying oil blends with a high concentration of these oils are the predominant oils currently used for deep-frying in North America, our primary goal was to complete development of a commercial FryCheck™ product for this market, then to examine its performance with other oil types.Many formulations were evaluated in order to provide the best response to oil polarity and to overcome early problems with a short shelf life.A major source of concern was ultimately found to be the acrylic acid in the adhesives commonly used for dry chemistry dipsticks. Ultimately, we elected to use two pieces of one-sided tape to hold the test pad onto the plastic backing. The test strip was widened to 1.0 cm for better appearance, handling characteristics, and adhesion of the pad to the strip. Test strips produced using better grade reagents were stable for several months at elevates (37°C) temperatures without a desiccant. Objective 2:Test Strip Validation:The %TPC results obtained using FryCheck™ were compared to results obtained using theOfficial Method Cd 20-91, "Determination of PolarCompounds in Frying Fats"method (AOCS Cd 20-91), a tedious laboratory method that is the reference standard for %TPC. We performed head-to-head comparison with other commercial "quick tests" for monitoring frying oil, including: 3M® Free Fatty Acid strips; Testo® Dielectric Constant Meter; OleoTest® and the Miroil Test. For most samples, the results show a good correlation of FryCheck visual readings with AOCS values, although FryCheck is presently less robust than desired. Objective 3: Develop FryCheck™ products that are optimized for additional fry oils:In order to provide for calibration and validation, a series of oil samples with known total polar content were prepared by collecting samples at various times during frying - worked very well.For product development and standardization calibrators are needed that can be frozen and used to perform quality control on test strip lots during manufacturing. Whereas FryCheck responded slower to hydrogenated soy oil, it responded more rapidly to Plenish than commodity soy. These results suggest that we may ultimately need to modify our formulation for optimal results with such oil types. Objective4:Determine the effect of food type on the performance of FryCheck™:The types of foods and the frying process in restaurants differ in many aspects from very controlled laboratory fry studies.Therefore, in addition to expanding the types of foods tested in our own lab, we collaborated with several outside organizations to evaluate how FryCheck performed for them. Oil samples were collected from the frying vats during the experiment and analyzed with FTNIR for total polar compounds content, then measured with FryCheck test strips. This work demonstrated that the deterioration of the oil could be followed using FryCheck test strips throughout the frying process. However, A pilot study performed at a very large agribusiness company showed that frying raw, uncoated/unseasoned chicken wings created a situation where rendering of the chicken fat was occurring. This caused the overall volume of the oil to increase during frying, which is usually opposite of what happens as cooks need to add or "top off" the oil during frying. FryCheck strips that were used to test this oil as the turned dark blue/grey, even when the oil was obviously unfit to consume, giving a false "good oil" response. We determined that the cause of this pad color anomaly was from the chicken fat which was effectively diluting the soybean oil. Objective5:Determine the utility of FryCheck™to detect adulterated "new" frying oil samples.In developing parts of the world where the demand for cooking oil is increasing, waste cooking oil is sometimes added to new oil and resold, creating a mixture termed "gutter oil" to increase profits. It is estimated that up to 10% of the total volume of cooking oil consumed in certain areas of China is gutter oil. The FryCheck™ dipstick is sensitive to the different composition of new oils, and in preliminary experiments we have observed that adulterating fresh vegetable oil with a small percentage of heavily used oil is detectable by using a FryCheck™ prototype. Objective 6:Develop and test a mobile phone application for more accurate pad color reading:Visual comparisons the %TPC to the color scale on the product vial are somewhat subjective and vary between individuals. Thus ,to achieve more accurate and precise readings of the %TPC values, we partnered with Spark Diagnostics, LLC. to adapt their proprietary, dipstick color reading technology to FryCheck™ strips. This technologyinvolves using the cell phone camera and calibrator colors incorporated into the dipstick for orientation and normalization of colors relative to the lighting conditions and analysis of the test results. The app was found to reproducibly measure color changes in the TPC dry chemistry dipstick to measure TPC in frying oil with the corresponding color change from dark blue (fresh) to bright yellow/orange (>26%).Additional 6 gray calibrator lines were printed at varying gray valuesfor the lighting conditions. Pattern recognition algorithms automatically correct for the tilt and other artifacts in the images such as blurriness due to user error. The standardized color response showed a linear correlation to the TPC concentration.Regression analysis to predict the TPC values for both iOS and Android systems showed a high predictive capability. The correlation coefficient >0.94 for all the models indicates a reliable prediction of TPC values.Further, the standard error of the prediction is in the range of 0.92 to 2.6% suggesting that the predictive values are much better than the color chart developed for visual readings of the dipstick (typically ~ +/- 6% units). Objective 7:Finalize high volume manufacturing, QA and packaging.The delamination of the pad from the surface of the plastic test strip has plagued this project from the start. We finally were able to eliminate tape containing acrylic acid and attach the test pad to the plastic backing by taping the top and bottom of the pad to the backing without adhesive on the back of the pad - resulted in a much more physically robust test that would not delaminate during shipping and handling. A short video tutorial is in preparation for uploading to thewww.frycheck.com. EPA Regulatory Compliance, Pre-Manufacture Notice:Not included in our original Work Plan is the submission of a Pre-Manufacture Notice (PMN) to the Environmental Protection Agency for our proprietary dye. The blue dye used as the indicator in FryCheck cannot be manufactured and sold to the public in the United States without being listed on the Toxic Substances Control Act (TSCA) Chemical Substance Inventory. In June 2021 we applied for a Pre-Manufacture Notice (PMN) to allow for the manufacturing of our dye in up to kilogram quantities.This applicationhas passed review by multiple EPA sections. However, as of this date, we are still awaiting final acceptance.Without this acceptance, we cannot make commercial quantities of the dye, nor are we permitted to have potential customers review our protype - except those few large organizations that have their own laboratory test kitchen.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: "FryCheckâ¿¢ - A New, Robust Quick Test for Frying Oil Quality". Enrique Martinez1, Denis Callewaert1, Bev Krejsa2, John Everard2 and Susan Knowlton3. 1Food Quality Testing Corp, Rochester Hills, Michigan, United States. 2Cortevaâ¿¢ Agriscience, Johnston, Iowa, United States. 3Cortevaâ¿¢ Agriscience, Wilmington, Delaware, USA. Presented at at the 17th International Congress of the European Federation for the Science and Technology of Lipids (EuroFedLipid) on 10/20 to 10/23, 2019, in Sevilla, Spain.

Progress 09/01/20 to 08/31/21

Outputs
Target Audience: Nothing Reported Changes/Problems:We returned back to normal operations in the lab with everyone working together in the building during the first week of June. Personnel had been working staggered shifts and days to reduce the chance of transmitting COVID. The major technical problem that overlapped this and the previous annual reports was the reactivity and degradation of the test strip pad due to the double-sided tape used to adhere it to the strip. In September 2020, after the tape was found to be the major problem, further formulation tweaking produced a strip that gave excellent color development with commodity soybean oil samples. We found that this problem affected the strips in a slow, progressive fashion, making their color responseless "vibrant" over time, but giving the same overall color change with oil calibrators. However, a problem with adhesion of the pad to the plastic strip arose where the top and bottom tape that adhered the pad from the front was difficult to handle as the pad would fall off of the strip. Doubling the width of the test strip solved this problem, and also made it easier to read and handle based on feedback from some of our collaborators. What opportunities for training and professional development has the project provided? Nothing Reported 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?Objective 1: Complete Development of FryCheck™ Test Strips for Commodity Soy and Canola Oils. Commodity canola oil is currently being used a frying study and will be evaluated the recently finalized FryCheck test strips. This will complete our initial set of important calibrators that are the most widely used oils for frying. Color Chart: A new color label scale will be prepared by matching the newly developed pad formulation. These colors are matched by eye using multiple lab personnel to a Pantone color library. Oil Additives: Using the finalized test strips, used oil samples will be spiked with samples of oil additives commonly used in frying. We will start with the levels that are used in new oil, and then make incremental increases in concentration until a noticeable change in pad color. Based on our experiences with other test strip chemistries, the levels of these oil additives will have to be several orders of concentration higher than what is used in new oil to show an appreciable effect on the performance of the pad. Objective 2:Test Strip Validation: We will run a second head-to-head evaluation between FryCheck and several frying oil quick tests, which include the following: the Testo® 270 Dielectric Constant Meter, 3M® Free Fatty Acid strips, and the OleoTest® liquid tests. This was performed two years ago using a separate formulation and test strip design that was further improved upon, thus it needs to be repeated. American Oil Chemists' Society (AOCS) Method Validation: After communicating with Scott Bloomer, the Director of Technical Services at the AOCS, we have decided to follow their advice and design a validation experiment for FryCheck. This will involve sending multiple AOCS-associated scientists a kit that contains ten unmarked frying oil samples with known TPC values and FryCheck strips. The tester will evaluate each unknown oil sample and report the data to OBR and the AOCS to determine if the test worked as described. Objective 3: Develop FryCheck™ products that are optimized for additional fry oils. Further formulation work will be done to optimize FryCheck for high oleic oils. Due to their inherent differences in chemical composition, with linoleic acid being the major fatty acid in commodity soy, an adjusted formulation will be prepared for optimal color change with High oleic (HO) soy. We will also use HO canola in a frying study and determine if the same strips can be used for this type of oil, as well. Objective 4:Determine the effect of food type on the performance of FryCheck. Three short frying studies will be performed with commodity soybean oil. All the studies will involve frying a fixed weight for each food type (i.e., 5.0 kg of fries, 5.0 kg of breaded chicken wings, 5.0 kg of puff pastry) in separate studies. The oils will then be collected and their AOCS values measured using the gold standard method, then their colors measured using the Ocean Optics reflectance meter and visual comparison. Finally, each oil will be measured multiple times with FryCheck and the differences in color change noted, thus showing the effect of food type on our test. Objective 5:Determine the utility of FryCheck™ to detect adulterated "new" frying oil samples. With the finalized test strip format and formulation, the dilution of this dialed-in formulation will be used to make strips that are more sensitive to oils that are adulterated with "gutter oil" that was prepared as described earlier. Objective 6:Develop and test a mobile phone application for more accurate pad color reading. Our subcontractor, PortaScience, is currently working on optimizing algorithms for reading the finalized test strip format. Further work will include populating the color database using FryCheck test strips that are used to test our library of fried oils and adding these colors and known TPC values into the app library to increase the accuracy of its readings. Also, the app itself will be worked on to looked more "polished" and easier to use. Our goal is to have a finished app within a years' time that can accurately give TPC values for soy and canola oils, and also tell the user whether or not the oil is good, should be used for a few more days, or should be discarded and changed immediately. Objective 7:Finalize high volume manufacturing, QA and packaging. A packaging expert has been contacted and given us some input as to who we can use to help us decide which packaging form is optimal for FryCheck. We are looking for guidance in help with picking out the correct heat impulse/vacuum sealed bag, and secondary packaging for multi-packs for distributorship. An important obstacle that needs to be addressed is the receipt of bulk-packed test strips from our manufacturer being sorted and packed in vials or bags of 15.

Impacts
What was accomplished under these goals? Formula and Test Strip Format Finalization: Objective 1. The pad formulation for FryCheck commodity soybean oil has been finalized, as have the design specifications for the test strip and the production process. Many formulations were evaluated, and with the problem of degradation due to tape adhesive solved, we were able to find a formulation that was stable at higher temperatures (45°C) for over a month. The test strip was widened from 0.5 cm to 1.0 cm improving the appearance, handling characteristics, and adhesion of the pad to the strip. Also, we made a change to our frying oil procedure to mimic more closely what happens in a restaurant setting. Previous frying studies did not top-off the oil during the frying experiment, but all restaurants add oil to their fryers when they get low. This does extend the length of frying experiments, but also better represents what happens in restaurants. Various Oil Type Frying Studies: Objective 3. We completed four additional types of oil: HO soy, commodity soy, peanut, and palm olein. The palm oil was more difficult to work with as it was solid at room temperature, and it turned almost black from frying puff pastry as this type of fat is used for donuts and other pastries. There are some restaurants that use a "creamy" frying fat, which affect how it interacts with the pad dye formulation. A creamy oil will be used in an upcoming frying study. Fortune 500 Company Frying Study: Objective 4. A study performed at a very large agribusiness company showed that frying raw, uncoated/unseasoned chicken wings created a situation where rendering of the chicken fat was occurring. This caused the overall volume of the oil to increase during frying, which is usually opposite of what happens as cooks need to add or "top off" the oil during frying. FryCheck strips that were used to test this oil as the turned dark blue/grey, even when the oil was obviously unfit to consume, giving a false "good oil" response. We determined that the cause of this pad color anomaly was from the chicken fat which was effectively diluting the soybean oil. Preparation of In-House Gutter Oil: Objective 5: Authentic samples of gutter oil proved very difficult to obtain. On October 24, 2020 Mrs. Shaw-Dore at the U.S. FDA China office was contacted for assistance with helping procure authentic gutter oil samples for our analysis. She replied: "We encourage you to contact an oil distributor, or a University that does research in the realm of food safety. They may be able to help you find someone that sells these products. The FDA cannot suggest any particular vendor. Nor can we provide any samples to you." This led us to Assistant professor Liu Xiaogang at the Singapore University of Technology and Design. He had been featured in Chemical and Engineering News by the American Chemical Society in an article that described his work with using fluorescence to detect adulteration in oils. I asked him where he obtained his gutter oil samples, and he said that they made them by heating oil for extended periods of time as well as cooking chicken. Next, Dinesh Kumar, PhD, Assistant Director Quality Assurance, was contacted to see if he would be interested in evaluating FryCheck. He works for the Food Safety and Standards Authority of India-FSSAI, Ministry of Health and Family Welfare-MoHFW, FDA Bhawan located in New Delhi. He replied that the test needs to be authenticated by a third-party such as the AOAC. We also contacted Sushama Kanetkar, Director at the Great Lakes St. Lawrence Governors & Premiers. Her address is: India Trade Office, Michigan Economic Development Corporation Affiliate Office Represented by SRK Affiliate 201, Infantry Court 130, Infantry Road in Bangalore. She was unable to find a source of adulterated oil for us to test but helped us gain a better understanding of the Indian edible oil market with a report that she compiled for us. Ultimately, we ended up preparing our own lot of "gutter oil" by cleaning a sample of oil from a large carboy that contained waste oil from several years' worth of frying studies. As there is no set, published procedure for cleaning this oil to use for adulteration, the oil was cleaned with solid absorbents, which partially decolorized and deodorized the material. This sample is ready to be made into dilutions with new oils and tested against our recently finalized formulation to determine if FryCheck can determine if new oil has been adulterated. Mylar Bags as Alternative to Vials: Objective 7. Most of this project has used polypropylene snap-top vials to hold 25 test strips as they are inexpensive, one-piece, and airtight. However, the strips can knock into each other during transport and handling, causing damage to the test pad. We evaluated a Mylar foil bag that can be heat impulse sealed under vacuum and is also resealable. The bags are also more appealing as they come in a variety of sizes, colors, and shapes. When compared to vials, bags can accept a larger label, be packed more densely, prevent the strips from moving when vacuum sealed, weigh less, and cost the same. We are still uncertain as to whether or not plastic outgassing is affecting the shelf life of our FryCheck strips, and as plastic bags contain less plastic than vials, they are a safer choice. High Oleic Soybean Oil: Objective 3. Corteva Agriscience has been partnering with since the start of the project. They are interested in using FryCheck to show how their high oleic soybean oil can be used longer than commodity soybean oil for frying and has other properties that are desirable for frying operations. However, after creating several iterations of the FryCheck formulation, high oleic and commodity soybean oils have shown to differ considerably in their chemical composition, thus giving different color responses with the test pads. We have suspected for some time that different oil types will interact with the pad dye formulation, and this comparison supports our theory. It may be necessary to create a different test strip for high oleic oils. EPA Regulatory Compliance, Pre-Manufacture Notice: Objective 7. The previous Progress Report was submitted while our Low Volume Exemption (LVE) was being processed by the EPA. However, due to technical aspects with processing the dye used in preparing FryCheck test strips, we were ultimately unable to obtain this exemption. We then withdrew our LVE application and filed for a Pre-Manufacture notice that allows us to address these issues more effectively. Without this, we are unable to synthesize the dye used in the FryCheck test strip pad formulation.

Publications

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:During the past year, FryCheck has been evaluated by 16 restaurants in Israel where it received overallfavorable reviews. These establishments stated that FryCheck would help them serve better quality food, and most of the users said it would help them use their oil longer and save them money. FryCheck was also used in an evaluation of a university dining hall fryer where it showed the degradation of their frying oils in several fryers.These types of establishments are both target customers for FryCheck as they are easy to approach and work with on a one-on-one basis. Changes/Problems:Indian Edible Oil Market Evaluation: Objective 3: Develop FryCheck™ products that are optimized for additional fry oils.Dr. Martinez contactedDr. Dinesh Kumar, PhD (AIIMS/IIT Delhi) Assistant Director (Tech) Quality AssuranceFood Safety and Standards Authority of Food Safety and Standards Authority of India.Ministry of Health and Family Welfare. Dr. Martinez inquired if the FSSAi would be interested in having their food scientists and inspectors evaluate FryCheck, and Dr. Kumar replied that the test needed further third-party validation, i.e. from theAssociation of Official Agricultural Chemists (AOAC). Dr. Martinez joined this organization and will inquire about having FryCheck undergo a similar third-party validation as this organization also has a method for determining total polar compounds in fats similar to the AOCS. Having third-party evaluations listed on our website and, potentially, on our packaging will give potential customers, including India, more confidence in purchasing FryCheck. Acquisition of Adulterated Oil: Objective 5: Determine the utility of FryCheck™ to detect adulterated "new" frying oil samples.After searching for sources of authentic adulterated oil samples either from China or India, we have decided to manufacture our own samples based on the advice from a professor in Singapore who has worked on this problem. Briefly, material from a grease trap will be obtained from one of our collaborating restaurantsand will be purified, deoderized and bleached, then mixed with new oil. This process will be a better representation of how adulterated oils are prepared,although authentic gutter oils would be preferred. Formulation Impurities: Objective 3: Develop FryCheck products that are optimized for additional fry oils. Recrystallizing the dye after it has been synthesized is a difficult process as it is very dark and visualizing the complete solubilization of the solids is not possible. Previous lots of dye have been recrystallized once in accordance with the literature, but this may not have been enough to remove most of the impurities. The dye was recrystallized a second time and found to perform better in the FryCheck formulation. The impurities have not been identified, but this additional purification step has shown us that the formulation is sensitive to foreign compounds. The cholesterol used in the formulatin was also changed to NF (National Formulary) grade from the 95% reagent grade which has been used throughout this project. This resulted in a much improved formulation requiring less solids per square centimeter in the pad. Test Strip Pad Delamination: Objective 7: Finalize high volume manufacturing, QA and packaging. After determining that the tape adhesive was reacting with our formulation, several new double-sided tapes were evaluated but not found to be any more effective in terms of pad adhesion. We later decided to discontinue using adhesive on the back of the pads and keep the glass matrix in place by taping the pad at the top and bottom on the front of the strip. Acid Sensitivity of Formulation: During several stability studies that were performed during this time, we found that the pad was being degraded by acids released by our oxygen scavenging packets, calcium chloride-based desiccant cards, and double-sided tape adhesive. All of these factors were found to be unecessary and further work with them was discontinued. Improved Dye and Formulation Sensitivity: Several additional dye variants were synthesized to see if they could extend the color change of high TPC level oils from yellow to red. However, after evaluating several new merocyanine dye derivatives, only one was found to behave similarly to the current dye. Also, several additives, including soluble multi-valent metals, surfactants and inert fillers were used to augment the reactivity of the formulation and reduce splotchyness of the resulting pad, but none succeeded. Glass Matrix Properties: The glass matrixused in preparing the test strips was thought to be interfering with the formulation by releasing sodium silicate into the impregnation solution during processing. However, several experiments, including pre-washing the glass matrix with water and chelates, silanizing the glass, drying the formulation at 100 degrees Celsius and pre-drying the glass to remove residual water did not have an appreciable effect on the stability of the pads. We now know that the degradation of these pads was more than likely due to the acidic nature of the double-sided tape used to prepare the strips. Several other glass matrices were evaluated from Ahlstrom-Munksjo, but were not found to work as well as the current Grade F matrix. What opportunities for training and professional development has the project provided?Dr. Martinez visited PortaScience in Moorestown, NJ in February to see how the strip manufacturing process was performed and to meet with the staff. During this visit, the coating, drying and mounting procedures were performed, with some improvements being made during the two runs that were performed during the visit. 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?National Restaurant Association Meeting 2021: Dr. Martinez and two other OBR employees will attend this conference in Chicago pending its status due to COVID. Fri, May 21, 2021 - Tue, May 25, 2021. The 2019 conference was cancelled due to the pandemic, and this would have been the official "rollout" of a FryCheck prototype to customers who were willing to take part in our first beta evaluation. Complete EPA Low Volume Exemption: To comply with 40 CFR 720.45 "Reviewing New Chemicals under the Toxic Substances Control Act (TSCA)" we filed a Low Volume Exemption with the Environmental Protection Agency and are expecting a response within three months. The reviewers may ask for more information on the properties of our compound, including toxicity data, physical properties, and others to finalize and accept the application. Once approved, this Exemption allows OBR to produce the blue dye used in the FryCheck formulation for our first consumer sales and distribution. Preliminary Commercial Evaluations: Thanks to our continued collaboration with Corteva™ Agriscience, we were introduced to food scientists and marketeers at a large agricultural food producer that is very interested in co-marketing FryCheck with their frying oils. They will be evaluating FryCheck in their test kitchens during the next few months. During a conversation with this group, and based on the outcome of their evaluation, we will be given the opportunity to evaluate FryCheck in at least one very large international fast food chain. AOCS/AOAC Third-Party Certification: Dr. Martinez has been corresponding with Scott Bloomer, Technical Services Director at the American Oil Chemists' Society, regarding the best way to have the AOCS examine FryCheck as a third-party validation. The AOCS will help contact certified oil testing labs and help us design an experiment showing the accuracy, reproducibility, robustness and correlation to the AOCS standard method for measuring total polar compounds. In-House Preparation of "Gutter Oil": Gutter oil is new oil which has been adulterated with purified waste oil collected from sewers, grease traps and other wastes, increasing the profit for the seller. It is a major problem in China and in parts of India, where regulations are more difficult to enforce. Dr. Martinez has contacted several people, including Professor Xiaogang Liu at the National University of Singapore. Prof. Liu was interviewed by Chemical and Engineering News from the American Chemical Society about work he has done with adulterated oils, which are hard to find here in the United States. In his email response, Professor Liu stated that their lab made their own adulterated oil samples as authentic samples are difficult to obtain. We decided that we would do the same and create our own gutter oil samples by obtaining grease trap clean-out material from one of our restaurant collaborators, purifying it, and mixing it with new oil to see what the limit of detection is with FryCheck strips.

Impacts
What was accomplished under these goals? EPA Regulatory Compliance: Objective 7: Finalize high volume manufacturing, QA and packaging. The blue dye used as the indicator in FryCheck cannot be manufactured and sold to the public in the United States without being listed on the Toxic Substances Control Act (TSCA) Chemical Substance Inventory. To satisfy this requirement, we applied for a Pre-Manufacture Notice Low Volume Exemption to allow for the manufacturing of our dye in kilogram quantities. This process was completed with assistance from our attorney at the Food Industry Council, LLC, Joel Chappelle on October 24th, with a response expected within 90 days. University Residence Hall FryCheck Evaluation: Objective 4: Determine the effect of food type on the performance of FryCheck™.Our collaborator Corteva™ Agrisciences evaluated our test strips against samples collected from Purdue University dining halls over a 16-day period. Oil samples were collected from the frying vats during the experiment and analyzed with FTNIR for total polar compounds content, then measured with FryCheck test strips. This work demonstrated that the deterioration of the oil could be followed using FryCheck test strips throughout the frying process. However, the actual fry cook could not directly evaluate FryCheck in the restaurant as this second phase of the experiment was cancelled due to the pandemic. Indian Oil Market: Objective 5:Determine the utility of FryCheck™ to detect adulterated "new" frying oil samples.Dr. Martinez consulted with Supriya Kanetkar, Director at the Great Lakes St. Lawrence Governors & Premiers, India Trade Office,Michigan Economic Development Corporation Affiliate Office. She is in the process of collecting information on the various types of oils which are used throughout the major parts of India. She has also offered help with identifying any agencies or inspectors that deal with adulterated oil and possibly attain some samples for our studies. She is also involved with introducing small businesses in Michigan to potential customers and distributors in India, which will help with introducting FryCheck to that continent. Improved Purity of Dye Formulation Reagents. Objective 1: Finalize the Development of FryCheck Test Strips for Commodity Soy and Canola oils. The dye, which is synthesized in-house, was purified using a second recrystallization, giving a formulation that used less dye. In addition to this, a higher purity cholesterol, National Formulary or NF Grade, was obtained, resulting in complete dissolution of the cholesterol with no residual solids as were visible as with reagent-grade cholesterol. To better solubilize the components, the main solvent, methanol, was changed to denatured ethanol, improving the solubility of the dye when preparing the formulation. These improvements resulted in an overall more sensitive pad when compared to previous iterations. Pad Chemistry Degradation from Acids: Objective 1: Finalize the Development of FryCheck Test Strips for Commodity Soy and Canola oils. Another problem which has affected the Frycheck test strips is the fading of the pad with time. During this past year, several iterations of the strips were prepared with varied tweaks to the formulations, and short-term stability studies were performed at elevated and room temperatures on strips prepared in-house. During these experiments, we discovered that the pad material that was not adhered to the strip failed at high temperatures in a different way than the pad which was laminated with the double-sided tape. A series of experiments was performed where the liquid formulation was placed into several test tubes and pieces of various double-sided tapes were added directly to the solution. Most of the tapes reacted with the solution, turning it from dark blue to yellow - a similar change that was observed when pads failed in the stability studies. It was discovered that acrylic acid and other carboxylic acids present in the acrylic adhesives in some tapes was causing the dye to change due to exposure to acid. These experiments have prompted us to use a process frequently employed in the lateral flow device field with taping the top and bottom of the pad to the backing without any adhesive on the back. This change effectively removes any degradation from acidic compounds in the double-sided tape adhesive and results in a much more secure pad. FryCheck™ Presented at EuroFedLipid Meeting: Objective 2: Test Strip Validation. Although it was not a proposed activity in the Phase II grant application, Dr. Martinez presented a postercomparing FryCheck™ to other commercially-available frying oil tests at the 17th International Congress of the European Federation for the Science and Technology of Lipids (EuroFedLipid) on 10/20 to 10/23, 2019, in Sevilla, Spain. Here, Dr. Martinez met with several oil scientists, manufacturers, and distributions, and gained a better understanding of the European edible oil market. Several of these scientists participate in the evaluation and validation of novel analytical methods and will be contacted when we are prepared for formal validation of our final product. Evaluation in Israeli Restaurants: Objective 1: Complete Development of FryCheck™ Test Strips for Commodity Soy and Canola Oils.Shai Medioni, Owner, Fandango Collection & Recycling LTD agreed to have 16 restaurants in Israel evaluate our test strips in their restaurants and give feedback based on a questionnaire that we prepared. Overall, the test was accepted and lauded by most of the users. All the restaurants said that the test would help their restaurant serve better food, keep using it if it were commercially available, would let them user their oil longer. 69% of the restaurants said that FryCheck would help them save money, and the same number said that they would pay $1.00 per strip, helping us to understand what our potential customers would consider to be a "fair price". Seven of these restaurants used soybean oil, three used sunflower and six used a blend purchased from Fandango. Other comments from the restaurants included their aproval of the test being "objective", ease of obtaining an oil sample from the fryer to test, and clarity of the instructions (translated to Hebrew). Improved Method for Mounting Pad: Objective 7: Finalize high volume manufacturing, QA and packaging.The delamination of the pad from the surface of the plastic test strip has plagued this project from the start. Recently, an improvement of the formulation was made, causing the pad's physical qualities to drastically change. The new method of taping the top and bottom of the pad to the backing without adhesive on the back of the pad resulted in a much more physically robust test that would not delaminate during shipping and handling. We determined that the new formulation was resulting in less solids being deposited in the glass fiber matrix, thus reducing the surface area available for the tape adhesive to flow into during lamination.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: "FryCheckÿ¢ÿ¿ÿ¢ - A New, Robust Quick Test for Frying Oil Quality". Enrique Martinez1, Denis Callewaert1, Bev Krejsa2, John Everard2 and Susan Knowlton3. 1Food Quality Testing Corp, Rochester Hills, Michigan, United States. 2Cortevaÿ¢ÿ¿ÿ¢ Agriscience, Johnston, Iowa, United States. 3Cortevaÿ¢ÿ¿ÿ¢ Agriscience, Wilmington, Delaware, USA. Presented at at the 17th International Congress of the European Federation for the Science and Technology of Lipids (EuroFedLipid) on 10/20 to 10/23, 2019, in Sevilla, Spain.

Progress 09/01/18 to 08/31/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Problems Encountered and Addressed: Concentrated Polar Blend Standards: Objective 3: Develop FryCheck products that are optimized for additional fry oils. In order to provide for calibration and validation, a series of oil samples with known total polar content are needed. Hence, near the end of Phase I of this project, we developed a series of used oil calibrators that were prepared by mixing concentrated polar fractions obtained from used oil with new oil. This process involved taking over-used oil samples and separating the polar component using silica gel and organic solvents. This polar material was then eluted from the silica gel to make a polar concentrate, which was then mixed with new oil to prepare oil samples with varied total polar compound content. A new pilot lot of strips was then manufactured that was formulated specifically to provide optimal results using these calibrators. After the strips were received, they were tested in-house at OBR using the same calibrators, and a portion of the strips were sent for evaluation by our collaborators at Corteva™ Agriscience. Although these strips performed as expected with the blended calibrators that we developed, they failed to differentiate among fresh oil samples with varying TPC content that were generated in frying studies conducted at Corteva™. These studies determined that blending new oil with a "polar concentrate" do not result in calibrators that represent a true "used" oil sample. Rather, the blended calibrators were likely enriched with saponified compounds that interfere with TPC measurements and cause a more drastic color change than would an actual used frying oil sample. The test strip formulation developed for these artificial blended calibrators produced a suboptimal color change when exposed to "normal" used oil samples. After this problem was identified, the formulation was again changed, by altering the concentration of PVP, to perform optimally with "real life" calibrator samples provided by Corteva™. After optimization using laboratory-made test strips, a new pilot lot of test strips was prepared at PortaScience. These strips were then evaluated at OBR and sent to Corteva™ to be re-evaluated. This change in the formulation - and the use of oil calibrators prepared by collecting samples at various times during frying - worked very well. To prepare additional calibrator samples needed for canola, soy, palm and their high-oleic acid equivalents, we constructed a small fry station to prepare calibrators in-house. Test Strip Pad Delamination: Objective 1: Complete development of FryCheck test strips for commodity soy and canola oils. The standard dimensions for commercial test strip pads (e.g. for testing urine samples or water testing) are 5mm by 5mm. We had initially made several hundred test strips with a 5.0 mm by 15 mm pad to make it easier to read the pad. However, after showing the strong color change achieved by our latest formulation to collaborators and a restaurant chain compliance manager, it was determined that a 5 by 5 mm pad should suffice, so we changed the dimensions for the test manufactured lot to the smaller size. However, the material that the pad is composed of is a laminated glass fiber that does not have the same robust handling characteristics as normal cellulose paper, so we encountered difficulties getting the smaller pads to maintain their integrity during manufacturing, shipping and handling. Therefore, we will be reverting to use of 5 x 15 mm pads in future production runs. Test Strip Pad Adhesion: Objective 1: Complete development of FryCheck test strips for commodity soy and canola oils. Technicians at PortaScience who manufacture test strips for us found that the glass paper adhered much better to the "belt" side of the paper adhered to the tape. This produced a strip with better adhesion to the plastic backing, resulting in less waste and in fewer detached pads when the strips were shipped to us. Test Strip Pad Drying Time: Objective 1: Complete development of FryCheck test strips for commodity soy and canola oils. PortaScience performed a series of experiments where the drying time of the test strip pad material (glass fiber) impregnated with the dye solution was passed through the tunnel dryer at several rates. It was determined from testing here at OBR that the pads that had a rate of 0.2 to 0.3 meters per minute showed the best color change, and the 0.3 meters per minute samples were then mounted and made into strips for further testing, considered to be our first large lot. Figure 3. Pantone CMYK color tabs were used to determine the FryCheck™ test strip color change. Figure 3. The fry station set up to generate oil calibrators. Obtaining Used Oil Samples for Calibrators: Objective 3: Develop FryCheck products that are optimized for additional fry oils. We have found it very difficult to obtain used oil samples with TPC content spanning the desired range from restaurants. There are no commercial sources for fry oil TPC calibrators and, as mentioned above, the blending of concentrated polar samples with fresh oil did not provide useful calibrators. For product development and standardization calibrators are needed that can be frozen and used to perform quality control on future test strip lots during manufacturing. After determining that we could not rely on restaurant chains to produce oil samples with different TPC content for us, let alone provide information on exactly how the oil was treated (types of food fried, fresh oil added, filtering, etc.), we decided that the best course was to set-up our own frying station (Figure 3). A Grindmaster-Cecilware EL15 stainless steel electric commercial countertop deep fryer with 15 lb. fry tank was purchased, along with two consumer stove vent hoods, as illustrated. A frying study using Idaho russet potatoes and chicken nuggets with commodity soybean oil is underway. Color-Matching Pads to Label Scale: Objective 6: Finalize high volume manufacturing, QA and packaging. Perhaps the most challenging aspect of this project during the past several months has been producing a color-comparison label (scale) for the test strip bottle that accurately matches the color of the test strips. Four color (CMYK: cyan, magenta, yellow and black) printing produces significantly variable results that are printer-specific, so that the first two lots of labels that we had printed by two different commercial printers were not acceptable - especially in the most critical range of 18-24% TPC. We are now exploring the use of PMS (Pantone color Matching System) colors that offer superior color-matching. The current color scale will use two CMYK (0% "New and 14% Good) and two PMS color patches (18% "Fair" and 24% "Poor), as the latter would not print correctly to the test strips using CMYK (Figure 3). The label on the right (Figure 4) Figure 4. The new, simplified FryCheck™ label. represents the fourth iteration of the color scale that has been refined to best match the test strip color change. Test Strip Vial: Objective 1: Finalize the development of FryCheck test strips for commodity soy and canola oils. During Phase I, we began investigating a vial that is lined with an integral desiccant. However, in this phase of the project we discovered that these specialized vials out-gasses an unknown volatile and have alkaline properties that caused the test strips to turn an off-orange color and ruin their functionality. An unlined, similar sized Capitol Vial did not stay shut during handling or with fluctuations in room temperature, but the design has since been improved to close better and will be used for stability studies. What opportunities for training and professional development has the project provided? Nothing Reported 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? Planned Activities: EuroFedLipid Conference: Dr. Martinez will attend the 17th EuroFedLipid Conference and Expo, October 20-23 in Sevilla, Spain and present a poster entitled, "FryCheck™ - A New, Robust Quick Test for Frying Oil Quality". Denis Callewaert as well as our collaborators John Everard and Susan Knowlton at Corteva™ Agriscience are co-authors on this poster which will discuss the current status of studies of the FryCheck™ dipstick. These include its performance in comparison to FTNIR, Oleotest, 3M Free Fatty Acid test strips and Testo methods, in measuring the TPC content and overall quality of high oleic soy and canola frying oils, as well as assay performance as a function of temperature. Since the TPC levels in frying oils are regulated in Europe, we believe that this conference will be highly productive and give us the opportunity to connect and work with regulators and potential customers that need our test in order to remain in compliance with frying oil regulations. The conference format includes a table-top display that will enable Dr. Martinez to demonstrate the performance of our test strips, but we will not hand out samples as they are not yet cleared by any regulatory agency for consumer use. EPA Premanufacture Notice: To comply with 40 CFR 720.45 "Reviewing New Chemicals under the Toxic Substances Control Act (TSCA)" we will be filing a Pre-Manufacture Notice with the Environmental Protection Agency. This process involves determining if FryCheck™ can be considered exempt from TSCA registration and be considered an "Article". Also, it will determine whether or not the dye used in the formulation needs any additional toxicity testing and classification, and/or registration with the EPA and/or a warning label and/or the creation of a Safety Data Sheet for FryCheck™. Preliminary Commercial Evaluations: Working with Corteva™ Agrisciences, we will have our first trial evaluation with the Purdue University dining halls system. This will involve collecting samples of frying oil from several campus cafeterias, testing them with laboratory-based oil analysis methods to find their TPC values, and then comparing them to FryCheck™ values. Based on these results, our collaborators will then provide selected restaurant owners several vials of our test strips for evaluation, accompanied by detailed instructions on how to run the test, the safety concerns regarding the test strips, and how to interpret the results. This evaluation process will include a detailed survey which will provide us with critical feedback on what potential end-user think of the test; including its ability to help them save money by less frequent fry oil changes and ease-of-use. Stability Study: With our latest lot of test strips, we will perform accelerated and long-term stability study at refrigerated, room, and elevated (40°C) temperatures and will last from the end of September 2019 to April 2020. This will also involve several bottles left open to the atmosphere to produce the worst-case scenario for strip storage. The results of this study will allow for us to assign an expiration date to the strips and understand their behavior over time. To determine the effects of long-term storage at elevated temperatures, the strips will be tested with oil calibrators at specific time points and compared to the current color scale. We have also devised a method for using our Ocean Optics reflectance spectrophotometer (utilized in Phase I of this grant) to measure the color intensities of specific wavelengths for each calibrator. We can then use these values to quantify any changes that occur in terms of color change in relation to degradation from storage. AOCS Evaluation: When the regulatory aspects of FryCheck™ have been understood and established, the first commercial lot will be manufactured. From this lot, samples will be submitted to the American Oil Chemists' Society, which will evaluate FryCheck™ and determine if FryCheck™ meets their requirements for approval as a semi-quantitative tool for monitoring frying oil degradation. AOCS certification would provide FryCheck™ with a higher level of credibility, allowing us to approach foreign markets with a globally-recognized third-party, industry specific validation. Figure 5. The new, simplified FryCheck™ label. Card Backing Evaluation: Several versions of white plastic backing that the test pads are mounted will be evaluated for ink adhesion. The current prototype strip with calibrator lines for use with the cell phone app, shown in Figure 5, has two sets of lines printed on it; the color calibrators which match the color that the strip turns when exposed to specific oil calibrators, and grayscale bands to correct for ambient lighting conditions. These colors will be printed in Pantone Color Matching System (PMS) colors identical to those used to create our current label. However, some plastics are difficult to print on compared to traditional papers as they have different ink adhesion characteristics. We are in contact with Tekra, which manufactures a wide variety of these plastic backings, and they are assisted us in picking several types of plastic with treatments for enhanced ink adhesion. We are also searching for printers that can print PMS colors on plastic films.

Impacts
What was accomplished under these goals? Problems Encountered and Addressed: Concentrated Polar Blend Standards:Objective 3: Develop FryCheck products that are optimized for additional fry oils. In order to provide for calibration and validation, a series of oil samples with known total polar content are needed. Hence, near the end of Phase I of this project, we developed a series of used oil calibrators that were prepared by mixing concentrated polar fractions obtained from used oil with new oil. This process involved taking over-used oil samples and separating the polar component using silica gel and organic solvents. This polar material was then eluted from the silica gel to make a polar concentrate, which was then mixed with new oil to prepare oil samples with varied total polar compound content. A new pilot lot of strips was then manufactured that was formulated specifically to provide optimal results using these calibrators. After the strips were received, they were tested in-house at OBR using the same calibrators, and a portion of the strips were sent for evaluation by our collaborators at Corteva™ Agriscience. Although these strips performed as expected with the blended calibrators that we developed, they failed to differentiate among fresh oil samples with varying TPC content that were generated in frying studies conducted at Corteva™. These studies determined that blending new oil with a "polar concentrate" do not result in calibrators that represent a true "used" oil sample. Rather, the blended calibrators were likely enriched with saponified compounds that interfere with TPC measurements and cause a more drastic color change than would an actual used frying oil sample. The test strip formulation developed for these artificial blended calibrators produced a suboptimal color change when exposed to "normal" used oil samples. After this problem was identified, the formulation was again changed, by altering the concentration of PVP, to perform optimally with "real life" calibrator samples provided by Corteva™. After optimization using laboratory-made test strips, a new pilot lot of test strips was prepared at PortaScience. These strips were then evaluated at OBR and sent to Corteva™ to be re-evaluated. This change in the formulation - and the use of oil calibrators prepared by collecting samples at various times during frying - worked very well. To prepare additional calibrator samples needed for canola, soy, palm and their high-oleic acid equivalents, we constructed a small fry station to prepare calibrators in-house. Test Strip Pad Delamination:Objective 1: Complete development of FryCheck test strips for commodity soy and canola oils.The standard dimensions for commercial test strip pads (e.g. for testing urine samples or water testing) are 5mm by 5mm. We had initially made several hundred test strips with a 5.0 mm by 15 mm pad to make it easier to read the pad. However, after showing the strong color change achieved by our latest formulation to collaborators and a restaurant chain compliance manager, it was determined that a 5 by 5 mm pad should suffice, so we changed the dimensions for the test manufactured lot to the smaller size. However, the material that the pad is composed of is a laminated glass fiber that does not have the same robust handling characteristics as normal cellulose paper, so we encountered difficulties getting the smaller pads to maintain their integrity during manufacturing, shipping and handling. Therefore, we will be reverting to use of 5 x 15 mm pads in future production runs. Test Strip Pad Adhesion:Objective 1: Complete development of FryCheck test strips for commodity soy and canola oils.Technicians at PortaScience who manufacture test strips for us found that the glass paper adhered much better to the "belt" side of the paper adhered to the tape. This produced a strip with better adhesion to the plastic backing, resulting in less waste and in fewer detached pads when the strips were shipped to us. Test Strip Pad Drying Time:Objective 1: Complete development of FryCheck test strips for commodity soy and canola oils.PortaScience performed a series of experiments where the drying time of the test strip pad material (glass fiber) impregnated with the dye solution was passed through the tunnel dryer at several rates. It was determined from testing here at OBR that the pads that had a rate of 0.2 to 0.3 meters per minute showed the best color change, and the 0.3 meters per minute samples were then mounted and made into strips for further testing, considered to be our first large lot. Figure 3. Pantone CMYK color tabs were used to determine the FryCheck™ test strip color change. Figure 3. The fry station set up to generate oil calibrators. Obtaining Used Oil Samples for Calibrators:Objective 3: Develop FryCheck products that are optimized for additional fry oils. We have found it very difficult to obtain used oil samples with TPC content spanning the desired range from restaurants. There are no commercial sources for fry oil TPC calibrators and, as mentioned above, the blending of concentrated polar samples with fresh oil did not provide useful calibrators. For product development and standardization calibrators are needed that can be frozen and used to perform quality control on future test strip lots during manufacturing. After determining that we could not rely on restaurant chains to produce oil samples with different TPC content for us, let alone provide information on exactly how the oil was treated (types of food fried, fresh oil added, filtering, etc.), we decided that the best course was to set-up our own frying station (Figure 3). A Grindmaster-Cecilware EL15 stainless steel electric commercial countertop deep fryer with 15 lb. fry tank was purchased, along with two consumer stove vent hoods, as illustrated. A frying study using Idaho russet potatoes and chicken nuggets with commodity soybean oil is underway. Color-Matching Pads to Label Scale:Objective 6: Finalize high volume manufacturing, QA and packaging.Perhaps the most challenging aspect of this project during the past several months has been producing a color-comparison label (scale) for the test strip bottle that accurately matches the color of the test strips. Four color (CMYK: cyan, magenta, yellow and black) printing produces significantly variable results that are printer-specific, so that the first two lots of labels that we had printed by two different commercial printers were not acceptable - especially in the most critical range of 18-24% TPC. We are now exploring the use of PMS (Pantone color Matching System) colors that offer superior color-matching. The current color scale will use two CMYK (0% "New and 14% Good) and two PMS color patches (18% "Fair" and 24% "Poor), as the latter would not print correctly to the test strips using CMYK (Figure 3). The label on the right (Figure 4) Figure 4.The new, simplified FryCheck™ label. represents the fourth iteration of the color scale that has been refined to best match the test strip color change. Test Strip Vial:Objective 1: Finalize the development of FryCheck test strips for commodity soy and canola oils. During Phase I, we began investigating a vial that is lined with an integral desiccant. However, in this phase of the project we discovered that these specialized vials out-gasses an unknown volatile and have alkaline properties that caused the test strips to turn an off-orange color and ruin their functionality. An unlined, similar sized Capitol Vial did not stay shut during handling or with fluctuations in room temperature, but the design has since been improved to close better and will be used for stability studies.

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