Crossbred beef steers (n = 30) finished on a corn silage ration were slaughtered in groups of 6 cattle per day at the University of Missouri red meats abattoir under USDA/FSIS inspection. Carcasses were chilled at 1 to 2°C for 48 h followed by fabrication where M. semimembranosus muscles (IMPS # 168; USDA, 2014) from one side of the carcass, also commonly referred to as top or inside round, were removed and closely trimmed to no visible external fat (IMPS #169A, USDA, 2014). Within each carcass, 2 sources of fat (kidney and pelvic from the carcass = KP, subcutaneous from the top round = S) were sourced to blend with the top round to achieve either 75 or 95% lean ground beef. Trim and fat blocks were individually ground through a coarse 10 mm plate (#8 Meat Grinder.35 HP, LEM Products, West Chester, Ohio). Final meat blocks (862 g lean and 45 g of fat) for 95% lean product and (680 g of meat and 227 g of fat) for 75% lean product were blended by hand and finely ground through a 4.5 mm plate. The 907 g of product from each treatment within animal was then used to create four, 115 g patties at each fat percentage (n = 60) to be used for the simulated retail display study. Each patty was formed using a patty press (Non-stick adjustable burger press, Model # 934, LEM Products, West Chester, OH). Patties were placed on 13.5 × 13.5 cm Styrofoam trays and overwrapped with oxygen permeable, polyvinyl chloride (UltraWrap Stretch Product #7021860 PVC #3, Anchor Packaging, St. Louis, MO; 15.500 to 16.275 cm³/m² per 24 h oxygen transmission rate at 23°C) and placed in simulated refrigerated retail storage (4°C; fluorescent bulbs; 48T21CWHO, Slyvania, Wilmington, MA; with a color temperature of 4200 K and a CRI of 60 for 24 h/d) where lipid oxidation was measured on d 1, 3, 5, and 7 of the study. The additional 447 g of sample was placed in a 11.4 × 22.9 cm Whirl-pak bag (Whirl-pak # S-16552, Nasco, Fort Atkinson, WI), stored at 4°C, and used for fat and moisture determination and fatty acid analysis. All laboratory analyses were conducted on fresh, never frozen ground beef samples to simulate the majority of ground beef sold at retail.

Extractable fat and moisture determination was performed on the day the meat was ground on each source and fat percentage combination (6 samples per animal) according to Keeton et al. (2003) using a CEM Moisture/Solids Analyzer and Smart Trac Rapid Fat Analysis system (CEM Corp., Matthews, NC). Briefly, 2 CEM square sample pads were placed into the moisture/solids analyzer, dried, and tared. After taring, 3.5 to 4.5 g of ground beef sample was then smeared across one of the pads. The second pad was placed on top of the sample, sandwiching the ground beef sample between the two sample pads, and moisture percentage was determined by weight using the CEM moisture/solids analyzer. Once the moisture analysis was completed, the dried sample and pads were rolled in Trac paper, placed into a CEM Trac tube, and packed tightly at the bottom. The tube was then placed in the CEM rapid fat analyzer where fat percentage was determined on dry basis using nuclear magnetic resonance and converted to wet basis. Each sample was run in triplicate and averaged for statistical analysis.

Methodology utilized for fatty acid quantification was an adaptation of the methods used by Folch et al. (1957) and Morrison and Smith (1964). All reagents were purchased from Fisher Scientific (Pittsburgh, PA). One g of sample was homogenized in a chloroform:methanol solution (CHCL3:CH3OH, 2:1, v/v) to extract the lipids. The sample was then filtered through a sintered glass filter funnel fitted with a Whatman 2.4 cm GF/C filter and 8 mL a solution of 0.74% KCl was added to the tube. The sample was allowed to sit for 2 h to separate the phases and then the upper phase was removed and discarded. The lower phase was then evaporated to dryness with nitrogen gas in a heated water bath at 70°C using a Meyer N-Evap Analytical Evaporator (Organomation Associates Inc., Berlin, MA). One mL of 0.5 N KOH in CH₃OH was added to the sample and the tube was placed in a water bath at 70°C for 10 min. Then, 1 mL of 14% boron trifluoride (BF3) in CH₃OH was added to the tube, flushed with nitrogen, loosely capped and placed in a water bath at 70°C for 30 min. After 30 min, the sample was cooled to room temperature and 2 mL of HPLC grade hexane and 2 mL of saturated NaCl was added. Next, the upper layer was removed and placed in a glass tube with approximately 800 mg of Na₂SO₄ to remove moisture from the sample. Following this, 2 mL of hexane was added to the tube with saturated NaCl and once more, the upper layer was removed and placed in the same tube with Na₂SO₄. The liquid portion was then transferred to a scintillation vial which was placed in a water bath at 70°C and the sample was evaporated with nitrogen. A Varian 420 gas chromatograph (Varian, Pala Alto, CA) was used to analyze fatty acid methyl esters; samples were injected onto a fused silica capillary column (SP– 2560; 100 m × 0.25 mm × 0.2 µm film thickness; Supelco, Bellefonte, PA). The temperature of the injector and of the flame-ionization detector was held constant at 240 and 260°C, respectively. Helium was used as the carrier gas at a constant pressure of 37 psi and the oven was operated at 140°C for 5 min (temperature programmed 2.5°C/min to 240°C and held for 16 min). Fatty acids were normalized meaning that the area of each peak was represented as a percentage of the total area. Iodine value (IV) was determined based on the equation described by AOCS (2017) Cd 1c-85 (2017): IV = (0.95 × C16:1) + (0.86 × C18:1n9) + (1.732 × C18:2n6) + (2.616 × C18:3n3) + (0.785 × C20:1). Iodine value is a measurement to estimate the amount of unsaturation of fatty acids found in carcass fat (DeRouchey et al., 2011). An internal standard fatty acid methyl ester was used and all fatty acid values are expressed as the percentage of fatty acids detected.

Lipid oxidation products were determined using the thiobarbituric acid reactive substances (TBARS) extraction method described by Pegg (2001). One of the 4 patties from each fat source and fat percentage combination of each animal was removed on d 1, 3, 5, and 7 of the shelf-life study to be used immediately (fresh) for lipid oxidation products determination. All reagents were purchased from Fisher Scientific (Pittsburgh, PA). Briefly, 5 g of each ground beef sample was weighed out and placed in a sample cup. 2.5 mL of an antioxidant solution, 50 mL of ice-cold TCA reagent, and 50 mL of distilled water was added to the sample in the cup and then homogenized for 3 min using a 2-speed Handheld Blender (Hamilton-Beach Model # 59760, Southern Pines, NC). The sample was then filtered and a 5-mL aliquot of sample solution was added along with 5 mL of thiobarbituric acid to a 50 mL conical centrifuge tube. The tubes were capped, vortexed, and heated in a boiling water bath for 35 min. The tubes were removed after 35 min from the water bath (Isotemp 110, Fisher Scientific), chilled in ice for 5 min to stop the reaction, and then the sample was transferred to an acrylic cuvette with a visible spectral range of 340 to 750 nm. Absorbance was measured at 532 nm using a spectrophotometer (Thermo spectronic Genesys 20 4001/4, Fisher Scientific). The TBARS values were obtained from the absorbance as described by Pegg (2001) and showed mg of malonaldehyde/kg of sample. Each sample was ran in duplicate and averaged for statistical analysis.

Statistical Analysis for proximate analysis and fatty acid profiles was performed using the MIXED procedure of SAS (Version 9.4, SAS Inst. Inc., Cary, NC) to obtain least square means and standard error estimates. The model included the fixed effects of fat source (KP and S) and fat percentage (5 and 25%) and all possible interactions. Objective color and TBARS were analyzed using the MIXED procedure of SAS and the model included the fixed effects of fat source, fat percentage, day (1, 3, 5, or 7), and all possible interactions. Significance was determined at P < 0.05.

Crossbred beef steers (n = 20) finished on a corn silage ration were slaughtered in groups of four cattle per day at the University of Missouri red meats abattoir under USDA/FSIS inspection. Carcasses were chilled at 1 to 2°C for 48 h followed by fabrication where M. semimembranosus muscles (IMPS #168, USDA, 2014) were removed and closely trimmed to no visible external fat (IMPS #169A, USDA, 2014). Within each carcass, 2 sources of fat, KP and S, were sourced to and processed and stored as described in Experiment 1 with the end result being final meat blocks of 95% lean product (969.6 g of meat and 51.0 g of fat) and 75% lean product (765.4 g of meat and 255.2 g of fat). The 1,020.6 g of product from each treatment within animal was then used to create eight, 113.4 g patties to be used for the shelf life study where samples were evaluated on d 1, 3, 5, and 7 to collect an instrumental measurement of color (L*, a*, b*), calculated myoglobin concentration, trained sensory panel, and TBARS. Aerobic plate counts were performed on the d 1 and 7 patties. The additional 113.4 g of sample was placed in a Whirl-pak bag (Whirl-pak # S-16552, Nasco), stored at 2°C, and used for fat and moisture determination. All laboratory analyses were conducted on fresh ground beef samples to simulate the majority of ground beef sold at retail.

Fat and moisture percentage determination was performed in triplicate as described in Experiment 1.

Surface color measurements (L*, a*, b*) of the ground beef patties were acquired utilizing a HunterLab MiniScan model 4500L (Hunter Associates Laboratory, Reston, VA) with a D65 light source, 1.27 cm aperture, geometry 45°/0° and physical standard was used to calibrate the MiniScan each day. Color coordinates were recorded on each sample’s specific day of removal from simulated retail display. On the first day of simulated retail display, all of the d 1 patties were measured prior to removal from the case for further analysis. This was continued on d 3, 5, and 7 of the study. Samples were evaluated in triplicate and averaged to achieve a more accurate representation of each ground beef patty.

Myoglobin concentrations of the ground beef samples were calculated via selected wavelengths described in the Meat Color Measurements Guidelines (American Meat Science Association; 2012). The reflectance was measured at the isobestic wavelengths 470, 530, 570, and 700 nm using HunterLab MiniScan model 45/0 LAV (Hunter Associates Laboratory, VA) in triplicate on d 1, 3, 5, and 7 of the study. The reflectance (R) was converted to reflex attenuance (A) using Eq. [1]. The A-values were then inserted into Eq. [2] to calculate MMb and into Eq. [3] to calculate deoxymyoglobin (DMb). Oxymyoglobin was then calculated using Eq. [4]:

Hue angle (HA), saturation index (SI), and a*/b* ratios were determined according to American Meat Science Association (2012). HA = [arctangent (b*/a*)] with larger values indicating less red, more MMb, and a more well-done cooked color (American Meat Science Association, 2012). Hue angle is very useful to indicate shifts in color over time toward discoloration. SI = (a*² + b*²)^1/2 with larger values indicating more saturation of the principal hue of the sample. Saturation index is very useful to indicate intensity of the hue is the product. Larger ratios of a*/b* indicate more redness and less discoloration (American Meat Science Association, 2012).

A total of 15 trained panelists participated in the ground beef sensory panel to determine odor and visual characteristics throughout shelf life of ground beef patties by methods described by Rhee et al. (1997) and Ohman et al. (2015). On d 1, 3, 5, and 7 of retail display, 6 to 8 panelists were asked to evaluate 16 patties. Patties were placed in a 15.24 cm diameter glass Petri dish and covered with a plastic watch glass. Each panelist evaluated all patties on retail display day they were present for. The patties were allowed to sit at room temperature (21°C) for 30 min, to develop and trap odor volatiles. To evaluate odor volatiles, panelist briefly lifted the watch glass and sniffed the patties. Off-odors and the intensity of the odor was immediately recorded, as described by Rhee et al. (1997). Putrid, sour, and fruity/sweet were the off-odor descriptors used on an eight point intensity scale (0 = no off-odor and 7 = extreme off-odor). Panelists completed a training session prior to the first evaluation that provided examples of odors and intensity levels. Strawberry yogurt was used as a reference for fruity/sweet odor and buttermilk was used to describe the sour off-odor. Odor intensity was described to panelists with the use of eight vials of increasing concentration of vanilla to distilled water (0 = 0% vanilla, 100% distilled water, 7 = 100% vanilla, 0% distilled water; Ohman et al., 2015). Odor and intensity references were provided at each evaluation. After odor analysis, watch glasses were removed and the patties were placed under a MacBeth light apparatus (Model EBX-22; 60W Incandescent bulb; Kollmorgan Corporation, Newburgh, NY) for evaluation of lean color and percent discoloration. Panelists were provided with references for lean color and percent discoloration at each evaluation. Lean color was determined using a scale as described by Montgomery et al. (2003), where 1 = dark brownish– greenish gray, 2 = light brownish– greenish gray, 3 = light gray, 4 = moderately dark red, 5 = slightly dark red, 6 = cherry red, 7 = moderately light cherry red, 8 = very light cherry red. Percent discoloration was determined on an 8 point scale (0 = no discoloration, 7 = complete discoloration). Procedures were approved by the University of Missouri Institutional Review Board.

Lipid oxidation products were determined using the TBARS extraction method in duplicate as described in Experiment 1.

Samples from each treatment were processed APC on d 1 and d 7. Duplicate 25 g portions of ground beef were weighed from 2 patties from each treatment for a total of four replications per treatment. Each 25-g sample was placed in separate sterile stomacher bag (Whirl-pak, Nasco) and homogenized in 225 mL of sterile 0.1% peptone water (BD Difco, Fisher Scientific) for 2 min (Seward Stomacher Model # 400C, Fisher Scientific, Pittsburgh, PA). Samples were serially diluted in sterile 0.1% peptone water at dilutions of 10^–1 to 10^–4 and 1 mL of each dilution was pour plated in approximately 20 mL of plate count agar. Plates were incubated at 5°C for 7 d. After the incubation period, colonies were manually counted, recorded, averaged and expressed as colony forming unit (CFU)/g. The minimum detection limit for determining APC was 10 CFU/g or 1 colony at 10^–1.

Statistical analysis for fat and moisture determination was performed using the MIXED procedure of SAS (Version 9.4, SAS Inst. Inc., Cary, NC) to obtain LS means and SE estimates. The model included the fixed effects of fat source (KP and S) and fat percentage (5 and 25%) and all possible interactions. Furthermore, statistical analysis for TBARS values, myoglobin percentages, color scores and sensory panel scores was performed using the repeated measures option in the MIXED procedure of SAS and the model included the fixed effects of fat source, fat percentage, d (1, 3, 5, or 7), and all possible interactions. Significance was determined at P < 0.05.