Progress 07/01/21 to 04/28/22

Outputs
Target Audience:We are focusing on the small- and medium-sized poultry farms that purchase their feed from an outside vender. Large-scale poultry farms make their own feed and go through hundreds of tons of feed per year, thus greatly reducing the chance of the feed going rancid. Smaller farms, from our understanding, tend to store their feed for longer periods of time. Our test can be used to help the farmer determine if this feed is suitable for feeding to their flock. It can also be helpful when purchasing large quantities of feed to determine how long it has been stored for and if it has gone rancid. Another potential market includes the feed producers who use large stores of grain to mill their feeds. These grains can sit for years before being sold and milled for feed, and can become rancid. We anticipate that, with further testing, that our instrument will help millers determine if their grain is too old for feed production. Changes/Problems:The most significant challenge and problem that was encountered during the project was the sensitivity variation in the TBARS and AOX strips. Previous work showed that these two test strip chemistries worked well for measuring analytes in urine and in other natural products and foodstuffs, including beverages, dog food, and many types of human foods. Several food types, including several cooking oils,were high enough in malonaldehyde that the color generated could be detected without an instrument and a color-comparison chart. This was the basis for this grant, as we anticipated that the poultry feeds would contain significant quantities of malonaldehyde due to their high surface area and non-ideal storage conditions. It was quickly discovered that, even with several different extraction methods, and with liquid phase testing, that the levels of malonaldehyde were very low in even aged feeds. We solved this issue by creating a fluorometric method for measuring the low levels of malonaldehyde in a liquid-based format. This method involves extracting the feed sample with water and then mixing with a pre-formulated TBARS indicator solution and allowing the two to react for five minutes. After this time, the sample was read using a prototype fluorometer that we fabricated and also with an analytical fluorometer, with both measurement methods correlating well. The advantage of using fluorometric reading vs colorimetric is that any other interfering compounds that would absorb at the same color as the TBARS reaction does not interfere with the fluorescent method, making it a much more selective and sensitive method. The new fluorometer prototype uses a 20 mL vial that is pre-filled with the TBARS indicator and then mixed with an equal volume of grain extract. The two are allowed to react for five minutes, and then a measurement is taken with the prototype. While not as simple and inexpensive as a test strip, this new method is much less subjective and produces more consistent results due to the nature of the analytical method. The antioxidant test strips showed the opposite problem as they were overloaded with the antioxidant concentrations that are added to the commercial chicken feeds. We found that, using a different chemical indicator that is similar to the CUPRAC indicator, that we could make a measurement colorimetrically. Thisindicator also has fluorescent properties that will need to be further optimized. This reaction would have to be done in a separate vial, and will require more development in Phase II of this project to optimize fluorescent parameters. The third major problem was the malfunctioning of the heated humidity chambers. We purchased a used chamber several months in advance of the grant start date to save money and to have the aged samples ready for testing. However, this unit malfunctioned and we decided to purchase a new unit from Memmert. This new unit started failing within several months, and only a few samples were able to be aged in that time. We ended up not using the unit after it failed for the third time (and after two service calls). This prevented us from performing a longitudinal study on the accelerated aging of poultry feed, with samples being taken at varied time points within the experiment. Some samples were then left out on the open atmosphere to age, while others were stored in a room temperature humidity chamber at approximately 80% RH. Fortunately, the samples that were aged in the Memmert did show significant chemical differences from the same samples that were stored at room temperature and humidity. 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? Nothing Reported

Impacts
What was accomplished under these goals? Major Phase I Achievements - Although dry chemistry test strip assays did not provide sufficiently sensitivity and range for point-of-use analysis of the freshness of poultry feeds, we demonstrated that our patented liquid phase fluorescent TBARS technology could provide a strong signal and reliable results. - We designed and fabricated a relatively crude but functional, easy-to-use, and inexpensive laboratory prototype fluorometer, using easily obtained components, that - in combination with our TBARS technology - can measure increases in rancidity as poultry feed ages. - Our inexpensive prototype fluorometer produces TBARS (rancidity) data that correlates well with our benchtop spectrofluorometer analytical instrument. As a direct cost-comparison, the original proposal would have required the customer to purchase a Waring blender for approximately $500 to produce samples that would be read using the proposed test strip assays. Our new alternative approach would require the purchase of a base unit fluorometer at a list price of $400 or less, and disposable integrated extraction/filtration/reaction chamber cartridge priced at approximately $20 each. This new approach would require the user to grind a weighed amount of feed in a coffee grinder and add it to the reaction cartridge. The total cost for this system falls within the same range as that the blender/test strip assay system. We are confident that our new approach to the antioxidant assay using a different colored indicator will also allow for an accurate determination of antioxidants in feed using the same base unit and test-specific disposable cartridges. - We evaluated an alternative chromophore for use in the CUPRAC assay and conducted experiments that support the feasibility of this fluorescence-based method for the rapid, point-of-use measurement of the antioxidant levels in poultry feeds that can be analyzed using the same test unit as the TBARS assay, just using a different disposable cartridge.

Publications

Progress 07/01/21 to 02/28/22

Outputs
Target Audience: Nothing Reported Changes/Problems:The original Proposal and Work Plan have been extensively modified to accommodate several technical challenges faced during the first months of this project. Using test strips to visually measure for rancidity and antioxidants in feed with a color chart has proved to be very difficult due to the minute and overwhelming levels of malonaldehyde and antioxidants present in chicken feeds, respectively. A more selective, accurate and precise method has been developed using an inexpensive fluorescence detector to measure rancidity (malonaldehyde) concentrations in feed. This new prototype produces data that correlates to our analytical benchtop fluorometer and will be optimized during the remainder of the project. The antioxidant capacity indicator was changed from neocuproine to bathocuproine, a compound that changes from light green/yellow to red in the presence of antioxidants. This new indicator can also be read fluorometrically and integrated into our prototype instrument. 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?With the prototype fluorometer assembled and working, the remainder of the project will focus on generating as much data as possible with our current collection of feed samples. The same samples will be extracted, and their peroxide value measured and correlated to the TBARS levels using two commercially available microplate assays for peroxides. Several of these samples will be extracted and the AOCS peroxide method will be run and correlated to the microplate results. The bathocuproine assay will be modified and attenuated to give measurable results with feeds containing high antioxidant levels.

Impacts
What was accomplished under these goals? Objective 1: Develop a feed sampling and homogenization protocol that is easy to use and produces a uniform sample for testing. Purpose: To finalize a method for feed sampling that produces the most reliable results for both assays. Problem Addressed: The end-user needs a reproducible and accurate method to sample feed for testing. Also, the sample needs to be homogenized in an extraction media to give a representative sample for both the TBARS and CUPRAC tests. Outcome: A volumetric study was performed using pellets, crumbles and whole grain feeds that showed up to 10% variability in the mass of feed per sample. This prompted us to exclude this sampling method and focus on measuring each sample using a balance. It may be possible for the farmer to measure the same type of feed volumetrically (i.e., if they consistently use the same type of feed) but the volumetric method cannot be used between different feed types. Objective 2: Demonstrate that dry chemistry CUPRAC and TBARS tests correlate directly with laboratory liquid phase assays. Purpose: To finalize an alternative method for measuring rancidity and antioxidant levels. Problem Addressed: The dry-format dipstick methods did not give acceptable results for comparison to the liquid phase assays, so this approach was abandoned. Outcome: The liquid phase TBARS assay was found to give acceptable results when read using a prototype fluorescence meter that has not been optimized. An alternative indicator was used in the CUPRAC assay that overcame issues with color interferences. Initial liquid-phase experiments failed show any formation of the TBARS pink chromogen in aged feeds when compared to fresh samples. Conversely, the antioxidant CUPRAC assay was overwhelmed by the high levels of antioxidants in the feeds. This led us to screen several different types of indicators that have been used for measuring antioxidants and rancidity, all of which could not be used in a test strip format. Our focus was returned to the liquid phase assays for both tests. The use of dry chemistry test strips to measure the freshness of poultry feed extracts is not sensitive enough for rancidity (TBARS) and must be attenuated for antioxidant (CUPRAC) due to the high levels of antioxidants added to most commercial feeds. However, the TBARS content of a feed sample can be measured using fluorescence in an inexpensive prototype. This method removes most interferences such as color contamination, reduces subjectivity from making an eyeball measurement, and gives a numerical readout. Bathocuproine will replace neocuproine in the antioxidant assay, as it forms a red color that can also be read fluorescently and works by the same chemical mechanism as the CUPRAC assay.

Publications